elife02384.xml

<?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN"  "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">02384</article-id><article-id pub-id-type="doi">10.7554/eLife.02384</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Biochemistry</subject></subj-group></article-categories><title-group><article-title>DNA polymerase V activity is autoregulated by a novel intrinsic DNA-dependent ATPase</article-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes" id="author-10913"><name><surname>Erdem</surname><given-names>Aysen L</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-10914"><name><surname>Jaszczur</surname><given-names>Malgorzata</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-10915"><name><surname>Bertram</surname><given-names>Jeffrey G</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-10916"><name><surname>Woodgate</surname><given-names>Roger</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="other" rid="par-4"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-4002"><name><surname>Cox</surname><given-names>Michael M</given-names></name><xref ref-type="aff" rid="aff4"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-10722"><name><surname>Goodman</surname><given-names>Myron F</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">Department of Biological Sciences</institution>, <institution>University of Southern California</institution>, <addr-line><named-content content-type="city">Los Angeles</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution content-type="dept">Department of Chemistry</institution>, <institution>University of Southern California</institution>, <addr-line><named-content content-type="city">Los Angeles</named-content></addr-line>, <country>United States</country></aff><aff id="aff3"><institution content-type="dept">Laboratory of Genomic Integrity</institution>, <institution>National Institute of Child Health and Human Development, National Institutes of Health</institution>, <addr-line><named-content content-type="city">Bethesda</named-content></addr-line>, <country>United States</country></aff><aff id="aff4"><institution content-type="dept">Department of Biochemistry</institution>, <institution>University of Wisconsin–Madison</institution>, <addr-line><named-content content-type="city">Madison</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Botchan</surname><given-names>Michael R</given-names></name><role>Reviewing editor</role><aff><institution>University of California, Berkeley</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>mgoodman@usc.edu</email></corresp><fn fn-type="con" id="equal-contrib"><label>†</label><p>These authors contributed equally to this work</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>24</day><month>04</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e02384</elocation-id><history><date date-type="received"><day>23</day><month>01</month><year>2014</year></date><date date-type="accepted"><day>28</day><month>03</month><year>2014</year></date></history><permissions><license xlink:href="http://creativecommons.org/publicdomain/zero/1.0/"><license-p>This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/publicdomain/zero/1.0/">Creative Commons CC0 public domain dedication</ext-link>.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife02384.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.02384.001</object-id><p><italic>Escherichia coli</italic> DNA polymerase V (pol V), a heterotrimeric complex composed of UmuD′<sub>2</sub>C, is marginally active. ATP and RecA play essential roles in the activation of pol V for DNA synthesis including translesion synthesis (TLS). We have established three features of the roles of ATP and RecA. (1) RecA-activated DNA polymerase V (pol V Mut), is a DNA-dependent ATPase; (2) bound ATP is required for DNA synthesis; (3) pol V Mut function is regulated by ATP, with ATP required to bind primer/template (p/t) DNA and ATP hydrolysis triggering dissociation from the DNA. Pol V Mut formed with an ATPase-deficient RecA E38K/K72R mutant hydrolyzes ATP rapidly, establishing the DNA-dependent ATPase as an intrinsic property of pol V Mut distinct from the ATP hydrolytic activity of RecA when bound to single-stranded (ss)DNA as a nucleoprotein filament (RecA*). No similar ATPase activity or autoregulatory mechanism has previously been found for a DNA polymerase.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02384.001">http://dx.doi.org/10.7554/eLife.02384.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.02384.002</object-id><title>eLife digest</title><p>DNA polymerases are enzymes that copy the genetic material within a cell using a strand of an existing double helix as a template to guide the synthesis of a new DNA strand. Since DNA is continuously exposed to damaging agents, and damaged DNA can derail the DNA replication machinery; a cell must either repair or bypass (via a process called translesion synthesis) this damage to copy its genome.</p><p>Most living things, from bacteria to humans, have specific DNA polymerases for translesion synthesis that can copy past damaged DNA, such as DNA Polymerase V in the bacterium <italic>E. coli</italic>. However, DNA polymerase V will also often introduce mistakes when it copies DNA that is not damaged—and cells will subsequently switch to use a different polymerase to accurately copy undamaged DNA.</p><p>DNA polymerase V is activated by binding to a protein called RecA and a molecule of adenosine triphosphate (ATP for short). ATP stores energy, which cells release by breaking down the molecule into simpler chemicals: but how do ATP and RecA work together to activate this polymerase?</p><p>Now, Erdem, Jaszczur et al. have addressed this question using biochemical techniques on purified polymerases, proteins and DNA fragments in a test tube. These experiments reveal that DNA polymerase V must bind to an ATP molecule before it can attach to the DNA template, and must remain bound to ATP while synthesizing the new DNA strand. After the activated polymerase has attached to the DNA, it will break down the molecule of ATP to free itself from the DNA. Furthermore, although the RecA protein can also break down ATP, Erdem, Jaszczur et al. found that a mutant RecA without this ability could still activate DNA polymerase V to break down this molecule itself.</p><p>Binding to and breaking down a molecule of ATP by a DNA polymerase has not been observed before as a method of directly regulating these enzymes' activity. Erdem, Jaszcuzur et al. suggest that, in living cells, this extra level of control would limit how long the DNA polymerase V spends attached to the DNA. As such, this polymerase would only be used to copy stretches of damaged DNA, but would not continue on to copy neighboring stretches of undamaged DNA where it would likely introduce new errors.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02384.002">http://dx.doi.org/10.7554/eLife.02384.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>DNA polymerase V</kwd><kwd>ATPase</kwd><kwd>RecA</kwd><kwd>pol V Mut</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd><italic>E. coli</italic></kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>ES012259</award-id><principal-award-recipient><name><surname>Erdem</surname><given-names>Aysen L</given-names></name><name><surname>Jaszczur</surname><given-names>Malgorzata</given-names></name><name><surname>Bertram</surname><given-names>Jeffrey G</given-names></name><name><surname>Goodman</surname><given-names>Myron F</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>GM21422</award-id><principal-award-recipient><name><surname>Erdem</surname><given-names>Aysen L</given-names></name><name><surname>Jaszczur</surname><given-names>Malgorzata</given-names></name><name><surname>Bertram</surname><given-names>Jeffrey G</given-names></name><name><surname>Goodman</surname><given-names>Myron F</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>GM32335</award-id><principal-award-recipient><name><surname>Cox</surname><given-names>Michael M</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution>NICHD/NIH Intramural Research Program</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Woodgate</surname><given-names>Roger</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>DNA synthesis by DNA polymerase V is regulated by an intrinsic DNA-dependent ATPase activity which has not been observed for any other polymerase.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>DNA polymerase V is a low fidelity TLS DNA pol with the capacity to synthesize DNA on a damaged DNA template (<xref ref-type="bibr" rid="bib22">Reuven et al., 1999</xref>; <xref ref-type="bibr" rid="bib29">Tang et al., 1999</xref>). It is encoded by the LexA-regulated <italic>umuDC</italic> operon and is induced late in the SOS response in an effort to restart DNA replication in cells with heavily damaged genomes (<xref ref-type="bibr" rid="bib14">Goodman, 2002</xref>). The enzyme is responsible for most of the genomic mutagenesis that classically accompanies the SOS response (<xref ref-type="bibr" rid="bib11">Friedberg et al., 2006</xref>).</p><p>RecA protein plays a complex role in the induction of pol V. As a filament formed on DNA (the form sometimes referred to as RecA*), RecA* facilitates the autocatalytic cleavage of the LexA repressor (<xref ref-type="bibr" rid="bib19">Little, 1984</xref>; <xref ref-type="bibr" rid="bib20">Luo et al., 2001</xref>). This leads directly to the induction of the SOS response. Some 45 min after SOS induction, those same RecA* filaments similarly facilitate the autocatalytic cleavage of UmuD<sub>2</sub> protein to its shorter but mutagenically active form UmuD′<sub>2</sub> (<xref ref-type="bibr" rid="bib4">Burckhardt et al., 1988</xref>; <xref ref-type="bibr" rid="bib21">Nohmi et al., 1988</xref>; <xref ref-type="bibr" rid="bib25">Shinagawa et al., 1988</xref>). UmuD′<sub>2</sub> then interacts with UmuC to form a stable UmuD′<sub>2</sub>C heterotrimeric complex (<xref ref-type="bibr" rid="bib31">Woodgate et al., 1989</xref>; <xref ref-type="bibr" rid="bib2">Bruck et al., 1996</xref>; <xref ref-type="bibr" rid="bib14">Goodman, 2002</xref>). UmuD′<sub>2</sub>C copies undamaged DNA and performs TLS in the absence of any other <italic>E. coli</italic> pol (<xref ref-type="bibr" rid="bib27">Tang et al., 1998</xref>), but only when RecA* is present in the reaction. UmuD′<sub>2</sub>C (<xref ref-type="bibr" rid="bib27">Tang et al., 1998</xref>; <xref ref-type="bibr" rid="bib17">Karata et al., 2012</xref>) or UmuC (<xref ref-type="bibr" rid="bib22">Reuven et al., 1999</xref>), have minimal pol activity in the absence of RecA*. Final conversion of the UmuD′<sub>2</sub>C complex to a highly active TLS enzyme requires the transfer of a RecA subunit from the 3′ end of the RecA* filament to form UmuD′<sub>2</sub>C-RecA-ATP, which we refer to as pol V Mut (<xref ref-type="bibr" rid="bib16">Jiang et al., 2009</xref>).</p><p>ATP plays an essential but heretofore enigmatic role in the activation process. Activation can proceed with ATP or the poorly-hydrolyzed analogue ATPγS. ATP is part of the active complex, with approximately one molecule of ATP per active enzyme (<xref ref-type="bibr" rid="bib16">Jiang et al., 2009</xref>). Under some conditions, activated and isolated pol V Mut exhibited polymerase activity only when additional ATP or ATPγS was added to the reaction (<xref ref-type="bibr" rid="bib16">Jiang et al., 2009</xref>). The function of the ATP complexed with pol V Mut is delineated in this report.</p></sec><sec id="s2" sec-type="results"><title>Results</title><p>Throughout this study, we utilize three variants of RecA protein for pol V activation. One is the wild-type (WT) RecA protein, which activates moderately in solution. The second is the RecA E38K/ΔC17 double mutant. The E38K mutation results in faster and more persistent binding of RecA protein to DNA, and the deletion of 17 amino acid residues from the RecA C-terminus eliminates a flap that negatively autoregulates many RecA activities (<xref ref-type="bibr" rid="bib18">Lavery and Kowalczykowski, 1992</xref>; <xref ref-type="bibr" rid="bib9">Eggler et al., 2003</xref>). The combination results in a RecA protein that activates pol V much more readily in vitro (<xref ref-type="bibr" rid="bib23">Schlacher et al., 2006</xref>; <xref ref-type="bibr" rid="bib16">Jiang et al., 2009</xref>). The third RecA variant is RecA E38K/K72R, combining the E38K change with the K72R mutation that all but eliminates the RecA* ATPase activity (<xref ref-type="bibr" rid="bib15">Gruenig et al., 2008</xref>).</p><sec id="s2-1"><title>ATP activation of pol V Mut</title><p>Pol V Mut can be formed and effectively isolated by incubating UmuD′<sub>2</sub>C complexes with RecA* that is bound to ssDNA oligonucleotides tethered to streptavidin-coated agarose beads, spinning the beads out of solution to remove RecA*, and taking the now active pol V Mut from the supernatant. In this initially described protocol (<xref ref-type="bibr" rid="bib16">Jiang et al., 2009</xref>), a small amount of ATP or ATPγS is transferred adventitiously from the supernatant with the pol V Mut. When WT RecA protein is used in this activation, the isolated pol V Mut WT is active only if supplemental ATP or ATPγS is added to the reaction mixtures (<xref ref-type="bibr" rid="bib16">Jiang et al., 2009</xref>). However, when a RecA variant that provides more facile activation of pol V was used, RecA E38K/ΔC17, the added ATP or ATPγS was apparently not needed for pol V Mut function (<xref ref-type="bibr" rid="bib16">Jiang et al., 2009</xref>). The reason for this disparity in the ATP requirement for pol V Mut function is resolved below.</p><p>To explore the role of ATP, an amended protocol (outlined in <xref ref-type="fig" rid="fig1">Figure 1A</xref>) was used that employed a spin column to more rigorously remove exogenous ATP or ATPγS after pol V Mut formation. As shown in <xref ref-type="fig" rid="fig1">Figure 1B</xref>, pol V Mut function now depends completely on added ATP or ATPγS when this activation protocol was utilized, regardless of which RecA variant was used in the activation. Pol V Mut is not activated by GTP, ADP or dTTP, (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1A</xref>) and does not incorporate ATP or ATPγS into DNA during synthesis (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1B</xref>). Thus, ATP or an ATP homolog is an absolute requirement for pol V Mut function. For pol V Mut WT, the addition of ATPγS supports synthesis, whereas ATP does not (<xref ref-type="fig" rid="fig1">Figure 1B</xref>). The same ATP effect was observed for pol V Mut WT synthesis on DNA containing an abasic site (<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>). dATP activation does not result in appreciable DNA extension (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). For pol V Mut E38K/K72R, synthesis is observed with either ATPγS, ATP or dATP (<xref ref-type="fig" rid="fig1">Figure 1B</xref>, <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). Pol V Mut E38K/ΔC17 can also use ATP, ATPγS or dATP as a required nucleotide cofactor (<xref ref-type="fig" rid="fig1">Figure 1B</xref>, <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). Notably, the requirement for ATPγS/ATP was completely masked in earlier studies of transactivation of pol V by RecA* filaments that remain in the solution with pol V Mut, because the ATPγS or ATP needed to form RecA* is always present in the transactivation reaction (<xref ref-type="bibr" rid="bib23">Schlacher et al., 2006</xref>).<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.02384.003</object-id><label>Figure 1.</label><caption><title>Pol V Mut requires ATP/ATPγS for activity.</title><p>Sketch of pol V Mut assembly; pol V is activated by RecA* bound to Cyanogen-Bromide Sepharose resin as described in ‘Materials and methods’. The pol V Mut assembly protocol ensures the separation of pol V Mut from free RecA, ssDNA and ATPγS. (<bold>B</bold>) Pol V Mut (400 nM) activity was detected on 5′-<sup>32</sup>P-labeled 3 nt oh HP (100 nM) in the presence or absence of ATP/ATPγS and dNTPs. To detect free RecA in the pol V Mut solution, ssDNA and ATPγS was added to the reaction. Comparable activity levels between ATPγS alone and ATPγS + ssDNA indicate that pol V Mut is intact and free of RecA.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02384.003">http://dx.doi.org/10.7554/eLife.02384.003</ext-link></p></caption><graphic xlink:href="elife02384f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02384.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Pol V Mut is not activated by GTP, ADP, or dTTP and does not incorporate ATP/ATPγS in to DNA during synthesis.</title><p>Pol V Mut WT, pol V Mut E38K/K72R, and pol V Mut E38K/ΔC17 were assembled according to the protocol in <xref ref-type="fig" rid="fig1">Figure 1A</xref>. (<bold>A</bold>) ATP, dATP, GTP, ADP, dTTP or ATPγS (500 μM) were used to activate pol V Mut for DNA synthesis and activity was checked in the presence of dNTPs (500 μM) and 3 nt oh HP (50 nM). (<bold>B</bold>) A HP containing TTT as its 3 nt oh was employed to determine if the various pol V Muts insert ATP or ATPγS during DNA synthesis. DNA extension is only observed in reactions where dATP is included. Other dNTPs are not present in any of the reactions.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02384.004">http://dx.doi.org/10.7554/eLife.02384.004</ext-link></p></caption><graphic xlink:href="elife02384fs001"/></fig><fig id="fig1s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02384.005</object-id><label>Figure 1—figure supplement 2.</label><caption><title>Pol V Mut WT activity on DNA containing an abasic site.</title><p>Pol V Mut (400 nM) activity was detected on 5′-<sup>32</sup>P-labeled 12 nt oh HP (100 nM), containing an abasic site 3 nts upstream from the 3′-OH, in the presence or absence of ATP/ATPγS and dNTPs. Lesion bypass is only observed when ATPγS is present.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02384.005">http://dx.doi.org/10.7554/eLife.02384.005</ext-link></p></caption><graphic xlink:href="elife02384fs002"/></fig></fig-group></p></sec><sec id="s2-2"><title>Pol V Mut is a DNA-dependent ATPase</title><p>Pol V Mut does not simply require ATP or ATPγS for activity; it possesses an intrinsic DNA-dependent ATPase activity (<xref ref-type="fig" rid="fig2">Figure 2</xref>). This is unprecedented for a DNA polymerase. A very sensitive ATPase assay, based on the fluorescence of a P<sub>i</sub> binding protein, is used in this work. The assay permits observation of the first 5 μM of ATP hydrolyzed, which is limited by the concentration of the fluorescent P<sub>i</sub> binding protein found in solution. A 30 nt ssDNA oligomer (1 μM) is present in all reactions. In <xref ref-type="fig" rid="fig2">Figure 2A</xref>, results are shown with pol V Mut made with WT RecA protein (pol V Mut WT). WT RecA protein alone exhibits limited stability on short oligonucleotides when present at sub-micromolar concentrations. Limited ATP hydrolysis by RecA WT alone is seen in this assay; the initial filaments produce a burst of ATP hydrolysis and then level off to a much slower rate, presumably reflecting filament binding, dissociation, and slow reassembly. Both phases of the reaction exhibit a dependence on RecA WT protein concentration (<xref ref-type="fig" rid="fig2">Figure 2A</xref>, <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1A</xref>). In contrast, after detectable free RecA protein and RecA* have been removed, equivalent concentrations of pol V Mut WT exhibit higher levels of ATP hydrolysis than do similar amounts of RecA alone (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). The P<sub>i</sub> release rate constant (<italic>k</italic><sub>cat</sub>) in the presence of ssDNA, 12 nt and 3 nt over hang (oh) Hairpin (HP) and in the absence of DNA are summarized in <xref ref-type="table" rid="tbl1">Table 1</xref>. For the calculation of rates see ‘Material and methods’.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.02384.006</object-id><label>Figure 2.</label><caption><title>Pol V Mut is a DNA-dependent ATPase.</title><p>(<bold>A</bold> and <bold>B</bold>) ATP hydrolysis by pol V Mut and RecA (0.1 μM, 0.2 μM and 0.4 μM each) was measured using MDCC-PBP (5 μM) in the presence of 30 nt ssDNA (1 μM) and ATP (500 μM). MDCC-PBP fluorescence increases as P<sub>i</sub> is released due to ATP hydrolysis. The measurements were taken at a resolution of 1 point per sec for approximately 1000 s. (<bold>C</bold>) Binding of 400 nM pol V, RecA and pol V Muts to etheno-ATP at varied concentrations was measured using rotational anisotropy. The error bars correspond to 1 SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02384.006">http://dx.doi.org/10.7554/eLife.02384.006</ext-link></p></caption><graphic xlink:href="elife02384f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02384.007</object-id><label>Figure 2—figure supplement 1.</label><caption><title>RecA WT and RecA E38K/K72R-dependent ATP hydrolysis.</title><p>ATP hydrolysis of (<bold>A</bold>) RecA WT and (<bold>B</bold>) RecA E38K/K72R was measured as a function of protein concentration in the presence of 1 µM 30 nt ssDNA and 500 µM ATP. P<sub>i</sub> release resulting from ATP hydrolysis was measured as a change in fluorescence of MDCC-PBP (5 μM). The measurements were taken at 1 point per sec resolution for approximately 1000 s.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02384.007">http://dx.doi.org/10.7554/eLife.02384.007</ext-link></p></caption><graphic xlink:href="elife02384fs003"/></fig></fig-group><table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.02384.008</object-id><label>Table 1.</label><caption><p>Pol V Mut and RecA ATP hydrolysis rate constants</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02384.008">http://dx.doi.org/10.7554/eLife.02384.008</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th/><th>ATPase <italic>k</italic><sub>cat</sub> (s<sup>−1</sup>)<xref ref-type="table-fn" rid="tblfn1">*</xref></th></tr></thead><tbody><tr><td colspan="2">pol V Mut WT</td></tr><tr><td> no DNA</td><td align="char" char="plusmn">(1.5 ± 0.1) × 10<sup>−3</sup></td></tr><tr><td> 12 nt oh HP</td><td align="char" char="plusmn">(9.0 ± 0.8) × 10<sup>−3</sup></td></tr><tr><td> 3 nt oh HP</td><td align="char" char="plusmn">(4.3 ± 0.1) × 10<sup>−3</sup></td></tr><tr><td> 30 nt ssDNA</td><td align="char" char="plusmn">(160 ± 5) × 10<sup>−3</sup></td></tr><tr><td colspan="2">RecA WT</td></tr><tr><td> 30 ssDNA</td><td align="char" char="plusmn">(100 ± 5) × 10<sup>−3</sup></td></tr><tr><td colspan="2">pol V Mut E38K/K72R</td></tr><tr><td> no DNA</td><td align="char" char="plusmn">(1.7 ± 0.2) × 10<sup>−3</sup></td></tr><tr><td> 12 nt oh HP</td><td align="char" char="plusmn">(4.4 ± 0.7) × 10<sup>−3</sup></td></tr><tr><td> 3 nt oh HP</td><td align="char" char="plusmn">(3.4 ± 0.7) × 10<sup>−3</sup></td></tr><tr><td> 30 nt ssDNA</td><td align="char" char="plusmn">(17 ± 1) × 10<sup>−3</sup></td></tr><tr><td colspan="2">RecA E38K/K72R</td></tr><tr><td> ssDNA</td><td align="char" char="plusmn">(0.6 ± 0.1) × 10<sup>−3</sup><xref ref-type="table-fn" rid="tblfn2">†</xref></td></tr><tr><td colspan="2">pol V Mut E38K/ΔC17</td></tr><tr><td> no DNA</td><td align="char" char="plusmn">(7.0 ± 1.5) × 10<sup>−3</sup></td></tr><tr><td> 12 nt oh HP</td><td align="char" char="plusmn">(54 ± 9) × 10<sup>−3</sup></td></tr><tr><td> 3 nt oh HP</td><td align="char" char="plusmn">(46 ± 2) × 10<sup>−3</sup></td></tr><tr><td> 30 nt ssDNA</td><td align="char" char="plusmn">(90 ± 10) × 10<sup>−3</sup></td></tr><tr><td colspan="2">RecA E38K/ΔC17</td></tr><tr><td> 30 nt ssDNA</td><td align="char" char="plusmn">(120 ± 15) × 10<sup>−3</sup></td></tr></tbody></table><table-wrap-foot><fn id="tblfn1"><label>*</label><p><italic>k</italic><sub>cat</sub> is an average of at least three independent measurements; ± SEM.</p></fn><fn id="tblfn2"><label>†</label><p><italic>k</italic><sub>cat</sub> was measured at 2.5 μM concentration for RecA E38K/K72R; ATP hydrolysis was not detectable at lower protein concentrations.</p></fn></table-wrap-foot></table-wrap></p><p>In principle, the ATPase properties of pol V Mut might be determined mainly, if not solely, by the properties of its RecA subunit. But that's in fact not the case. To address whether the pol V Mut -associated ATPase activity can be distinguished from the intrinsic DNA-dependent ATPase activity of RecA, we assembled pol V Mut with a RecA (E38K/K72R) mutant deficient in ATPase activity (<xref ref-type="bibr" rid="bib15">Gruenig et al., 2008</xref>). Using the same highly sensitive fluorescence-based assay as described for panel 2A, we were unable to detect DNA-dependent ATPase activity for RecA E38K/K72R above background (<xref ref-type="fig" rid="fig2">Figure 2B</xref>). However, in contrast, pol V Mut E38K/K72R exhibits substantial ATPase activity. The observed ATPase scales with the concentration of pol V Mut E38K/K72R (<xref ref-type="fig" rid="fig2">Figure 2B</xref>), as is also the case for pol V Mut WT (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). Therefore, pol V Mut is a DNA-dependent ATPase (<xref ref-type="fig" rid="fig2">Figure 2</xref>). As RecA E38K/K72R protein hydrolyzes little or no ATP on its own (<xref ref-type="fig" rid="fig2">Figure 2B</xref>, <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1B</xref>), the pol V Mut activity cannot be attributed to a low level of contamination by RecA protein. A small amount of ATP hydrolysis above background can be observed with RecA E38K/K72R by increasing its concentration to 2.5 μM (84 nM Pi release/min, <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1B</xref>).</p><p>We previously reported that the active complex of pol V Mut is composed of pol V-RecA-ATP (<xref ref-type="bibr" rid="bib16">Jiang et al., 2009</xref>). We can further distinguish pol V Mut from either pol V or free RecA in an assay measuring binding to etheno-ATP via anisotropy (<xref ref-type="fig" rid="fig2">Figure 2C</xref>). Pol V Mut made with either RecA WT or RecA E38K/K72R registers a substantial and concentration-dependent signal in this assay, while binding of etheno-ATP to either pol V or RecA alone is much weaker.</p></sec><sec id="s2-3"><title>Pol V Mut requires ATP/ATPγS to bind p/t DNA</title><p>To further explore the requirement for the addition of a ribonucleotide cofactor, we measured pol V Mut binding to p/t DNA and DNA synthesis as a function of the concentration of added ATPγS or ATP (<xref ref-type="fig" rid="fig3">Figure 3</xref>). Binding is absolutely dependent on a nucleotide cofactor. Pol V Mut WT binding to p/t DNA with a 3 nt template overhang (3 nt oh) increases roughly linearly up to about 180 μM ATPγS, saturating at about 220 μM (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). However, binding is not detectable in the presence of ATP. Increasing the length of the template overhang to 12 nt has essentially no effect on binding as a function of nucleotide concentration (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). However, with ATPγS pol V Mut binds with higher affinity to the p/t DNA 12 nt oh (<xref ref-type="table" rid="tbl2">Table 2</xref>). DNA synthesis corresponds closely to ATPγS-dependent pol V Mut WT binding, showing a linear increase in primer extension up to about 180 μM ATPγS, reaching about 70% total primer usage at about 500 μM ATPγS (<xref ref-type="fig" rid="fig3">Figure 3B</xref>). There is no primer extension with ATP (<xref ref-type="fig" rid="fig3">Figure 3B</xref>). Pol V Mut E38K/K72R binds to p/t DNA with ATPγS (<xref ref-type="fig" rid="fig3">Figure 3C</xref>; <xref ref-type="table" rid="tbl2">Table 2</xref>). Although it does not appear to bind DNA appreciably in the presence of ATP in this assay, some binding must occur as DNA synthesis is clearly observed (<xref ref-type="fig" rid="fig3">Figure 3D</xref>).<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.02384.009</object-id><label>Figure 3.</label><caption><title>Pol V Mut binding and activity as a function of nucleotide.</title><p>Binding of pol V Mut WT (1 μM) (<bold>A</bold>), pol V Mut E38K/K72R (400 nM) (<bold>C</bold>), and pol V Mut E38K/ΔC17 (400 nM) (<bold>E</bold>) to 3 nt oh HP was measured as a change in rotational anisotropy. Activity of pol V Mut WT (400 nM) (<bold>B</bold>), pol V Mut E38K/K72R (400 nM) (<bold>D</bold>), and pol V Mut E38K/ΔC17 (400 nM) (<bold>F</bold>) was quantified on 5′-<sup>32</sup>P-labeled 3 nt oh HP with varying concentrations of nucleotide and 500 μM dNTPs. A gel showing primer utilization (%PU) as a function of nucleotide is presented to the right of the graph. ATP (open circle) and ATPγS (filled circle). The error bars correspond to 1 SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02384.009">http://dx.doi.org/10.7554/eLife.02384.009</ext-link></p></caption><graphic xlink:href="elife02384f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02384.010</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Pol V Mut binding to 12 nt oh HP DNA as a function of ATP/ATPγS.</title><p>Binding of (<bold>A</bold>) pol V Mut WT (<bold>B</bold>) pol V Mut E38K/K72R, and (<bold>C</bold>) pol V Mut E38K/ΔC17 (400 nM each) to fluorescein-labeled 12 nt oh HP (50 nM) was measured as a function of ATP (open circles) and ATPγS (filled circles). DNA binding was observed as a change in rotational anisotropy. The error bars correspond to 1 SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02384.010">http://dx.doi.org/10.7554/eLife.02384.010</ext-link></p></caption><graphic xlink:href="elife02384fs004"/></fig><fig id="fig3s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02384.011</object-id><label>Figure 3—figure supplement 2.</label><caption><title>Pol V Mut WT activity as a function of ATPγS concentration in the presence of ATP.</title><p>Pol V Mut WT (400 nM) DNA synthesis is measured as a function of ATPγS (filled circles) on 3 nt oh HP (50 nM). When ATP (500 μM) is present at each ATPγS concentration, primer utilization of pol V Mut WT is decreased (open circles) compared to reactions where no ATP was included. The error bars correspond to 1 SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02384.011">http://dx.doi.org/10.7554/eLife.02384.011</ext-link></p></caption><graphic xlink:href="elife02384fs005"/></fig></fig-group><table-wrap id="tbl2" position="float"><object-id pub-id-type="doi">10.7554/eLife.02384.012</object-id><label>Table 2.</label><caption><p>Pol V Mut affinity to 12 nt oh HP DNA</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02384.012">http://dx.doi.org/10.7554/eLife.02384.012</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th rowspan="2">Pol V Mut</th><th align="center" colspan="2">K<sub>d</sub> (nM)</th></tr><tr><th>ATP</th><th>ATPγS</th></tr></thead><tbody><tr><td>pol V Mut WT</td><td><italic>nb</italic></td><td align="char" char="plusmn">876 ± 52</td></tr><tr><td>pol V Mut E38K/K72R</td><td><italic>nb</italic></td><td align="char" char="plusmn">920 ± 112</td></tr><tr><td>polV Mut E38K/<bold>Δ</bold>C17</td><td>312 ± 46</td><td align="char" char="plusmn">469 ± 27</td></tr></tbody></table><table-wrap-foot><fn><p>nb–binding not detected.</p></fn></table-wrap-foot></table-wrap></p><p>The same experiments performed with pol V Mut E38K/ΔC17 show that binding and activity correlate well with ATPγS and ATP. Here, a much higher affinity to p/t DNA with ATP (<xref ref-type="table" rid="tbl2">Table 2</xref>) allows binding and primer extension (17% at 750 μM ATP, <xref ref-type="fig" rid="fig3">Figure 3E,F</xref>). Pol V Mut E38K/ΔC17 requires less ATPγS (78% at 100 μM ATPγS) for optimal binding and activity compared to the other pol V Muts, corresponding to the more robust activation of pol V consistently seen with this RecA variant (<xref ref-type="bibr" rid="bib23">Schlacher et al., 2006</xref>; <xref ref-type="bibr" rid="bib16">Jiang et al., 2009</xref>).</p><p>The data in <xref ref-type="fig" rid="fig3">Figure 3</xref> establish an absolute requirement for an ATP cofactor for pol V Mut binding to p/t DNA. In no case is DNA binding detected in the absence of ATP or an ATP homolog. Since binding is a precursor to synthesis, the same nucleotide requirement holds for pol V Mut-catalyzed primer extension (<xref ref-type="fig" rid="fig1">Figures 1B</xref>, <xref ref-type="fig" rid="fig3">Figure 3</xref>). The link that's missing is the role of ATP hydrolysis in the DNA binding/synthesis process. Indirect evidence hinting at just such a link is the observation that pol V Mut WT hydrolyzes ATP about 9-fold more rapidly on ssDNA than pol V Mut E38K/K72R and about 2-fold more rapidly than pol V Mut E38K/ΔC17 (<xref ref-type="table" rid="tbl1">Table 1</xref>), suggesting perhaps that the more rapid ATP hydrolysis by pol V Mut WT diminishes its ability to remain bound to DNA long enough to catalyze synthesis under these in vitro conditions. Pol V Mut WT-dependent primer extension significantly decreases with a mixture of ATP/ATPγS (<xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2</xref>), further suggesting that the fraction of enzyme that binds and hydrolyzes ATP is not associated with DNA long enough to catalyze appreciable primer elongation. With p/t DNA, ATP hydrolysis is slower for pol V Mut E38K/K72R compared to pol V Mut WT (<xref ref-type="table" rid="tbl1">Table 1</xref>), which could account for incorporation with added ATP. In the case of pol V Mut E38K/ΔC17, although ATP hydrolysis is more rapid on p/t DNA compared to pol V Mut WT (<xref ref-type="table" rid="tbl1">Table 1</xref>), binding is much tighter (<xref ref-type="table" rid="tbl2">Table 2</xref>), which could plausibly explain primer extension observed with added ATP.</p></sec><sec id="s2-4"><title>ATP hydrolysis releases pol V Mut from p/t DNA</title><p>To obtain direct evidence for a possible role of ATP hydrolysis in the pol V Mut reaction pathway, we used pol V Mut formed with RecA E38K/ΔC17, which binds p/t DNA with higher affinity than either pol V Mut WT or pol V Mut E38K/K72R (measured K<sub>d</sub> for pol V Mut E38K/ΔC17 = 469 nM, compared to 876 nM for pol V Mut WT; <xref ref-type="table" rid="tbl2">Table 2</xref>). There is a concomitant ∼5 to 10-fold greater DNA-dependent ATPase activity for pol V Mut E38K/ΔC17 compared with pol V Mut WT (<xref ref-type="table" rid="tbl1">Table 1</xref>). The pol V Mut E38K/ΔC17-p/t DNA complex formed in the presence of ATP was stable for a long enough time to conveniently monitor its dissociation (<xref ref-type="fig" rid="fig4">Figure 4</xref>). Fluorescent-labeled p/t DNA (12 nt oh, 50 nM) has a rotational anisotropy (RA) of 0.05 when diffusing freely in solution (<xref ref-type="fig" rid="fig4">Figure 4</xref>). When incubated in the presence of eightfold excess pol V Mut E38K/ΔC17 (400 nM), essentially all of the DNA is bound in a complex with pol V Mut (RA = 0.12). Immediately following complex formation (t ∼ 0), 160-fold excess unlabeled p/t DNA (8 μM) was added to trap pol V Mut as it dissociates. Pol V Mut E38K/ΔC17 forms a stable complex with p/t DNA in the presence of ATPγS, remaining bound for at least 4 min without dissociating. In contrast, when the complex is formed with ATP, pol V Mut E38K/ΔC17 dissociates exponentially as a function of time, asymptotically reaching ∼100% free DNA at about 75 s (<xref ref-type="fig" rid="fig4">Figure 4</xref>). The off-rate determined as the first-order rate constant is 0.053 s<sup>−1</sup>. The magnitude of the off-rate is in remarkably close agreement with the DNA-dependent ATPase single-turnover rate constant for pol V Mut E38K/ΔC17 = 0.054 s<sup>−1</sup> (<xref ref-type="table" rid="tbl1">Table 1</xref>). The data strongly suggest that ATP hydrolysis is responsible for pol V Mut-p/t DNA dissociation. This can also be seen during DNA synthesis, where longer segments of DNA are synthesized with ATPγS than with a similar concentration of ATP (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>). The correspondence of dissociation rate to ATP hydrolysis rate further suggests that perhaps a single ATP turnover is sufficient to trigger dissociation of pol V Mut from a 3′-OH primer end. The absence of dissociation in the presence of the weakly hydrolyzed ATPγS strongly reinforces this conclusion. Pol V Mut WT and pol V Mut E38K/K72R, act similarly to pol V Mut E38K/ΔC17, remaining stably bound to p/t DNA in the presence of ATPγS (<xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2</xref>). Although ATP hydrolysis affects pol V Mut–DNA complex stability, the integrity of the pol V Mut protein complex is not affected by hydrolysis, with RecA remaining bound to UmuD′<sub>2</sub>C (<xref ref-type="fig" rid="fig4s3">Figure 4—figure supplement 3A,B</xref>).<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.02384.013</object-id><label>Figure 4.</label><caption><title>Pol V Mut E38K/ΔC17 off-rate in the presence of ATP and ATPγS.</title><p>Fluorescence depolarization of fluorescein-labeled (12 nt oh HP) DNA was used to measure the dissociation constant of pol V Mut E38K/ΔC17 in the presence of ATP (filled circles) and ATPγS (open circles). A stable protein–DNA complex (400 nM and 50 nM, respectively) was preformed in the presence of nucleotide followed by the addition of excess (160 times) trap DNA (unlabeled 12 nt oh HP). The decrease in anisotropy over time was fit to an exponential decay to determine <italic>k</italic><sub>off</sub> (0.053 ± 0.0025 s<sup>−1</sup>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02384.013">http://dx.doi.org/10.7554/eLife.02384.013</ext-link></p></caption><graphic xlink:href="elife02384f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02384.014</object-id><label>Figure 4—figure supplement 1.</label><caption><title>Pol V Mut E38K/ΔC17 primer extension length as a function of ATP or ATPγS concentration.</title><p>DNA extension was measured with varying concentrations of ATP (left panel) and ATPγS (right panel) on 12 nt oh HP to determine the lengths of primer synthesized as a function of NTPs. The unextended primer is indicated as HP and the addition of a nucleotide is marked as nt 1–4 and nt 12 in the center of the panels. The length of DNA synthesized by pol V Mut E38K/ΔC17 increases with the concentration of ATP/ATPγS. Pol V Mut E38K/ΔC17 is far more active with ATPγS compared with ATP, which is consistent with in inability to dissociate from p/t DNA in the absence of ATP hydrolysis.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02384.014">http://dx.doi.org/10.7554/eLife.02384.014</ext-link></p></caption><graphic xlink:href="elife02384fs006"/></fig><fig id="fig4s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02384.015</object-id><label>Figure 4—figure supplement 2.</label><caption><title>Dissociation of pol V Mut WT and pol V Mut E38K/K72R in the presence of ATPγS.</title><p>(<bold>A</bold>) pol V Mut WT and (<bold>B</bold>) pol V Mut E38K/K72R (400 nM) were pre-bound to fluorescein-labeled 12 nt oh HP (50 nM) in the presence of ATPγS. Excess trap DNA (8 μM unlabeled 12 nt oh HP) was added to the stable protein–DNA complex and DNA binding of pol V Mut was monitored as a change in rotational anisotropy (circles). Fluorescein-labeled 12 nt oh HP alone is indicated as a black line.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02384.015">http://dx.doi.org/10.7554/eLife.02384.015</ext-link></p></caption><graphic xlink:href="elife02384fs007"/></fig><fig id="fig4s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02384.016</object-id><label>Figure 4—figure supplement 3.</label><caption><title>Pol V Mut remains intact in the presence of ATP/ATPγS and during DNA synthesis.</title><p>RecA<sub>F21AzF</sub>-Alexa Fluor 488 was used to form RecA* during pol V Mut assembly resulting in the formation of fluorescently-labeled pol V Mut (pol V Mut WT<sub>F21AzF</sub>-Alexa Fluor 488). (<bold>A</bold>) The rotational anisotropy of pol V Mut WT<sub>F21AzF</sub>-Alexa Fluor 488 (100 nM) was measured under ATP hydrolysis and DNA synthesis conditions (‘Materials and methods’). No change was observed upon addition of ATP/ATPγS, 12 nt oh HP (1 μM) or during DNA synthesis (2, 5 and 10 min) indicating that pol V Mut remains intact. The anisotropy of free RecA<sub>F21AzF</sub>-Alexa Fluor 488 (100 nM) was measured in parallel. The error bars correspond to 1 SEM. (<bold>B</bold>) DNA synthesis was measured for pol V Mut WT and pol V Mut WT<sub>F21AzF</sub>-Alexa Fluor 488 demonstrating that intact enzyme extends DNA under experimental conditions (pol V Mut: 100 nM and HP 1 μM) at the time periods (2, 5, 10, 20, and 30 min) used for rotational anisotropy measurements (<bold>A</bold>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02384.016">http://dx.doi.org/10.7554/eLife.02384.016</ext-link></p></caption><graphic xlink:href="elife02384fs008"/></fig></fig-group></p><p>The failure of pol V Mut WT to synthesize DNA in the presence of ATP can presumably be attributed to its inability to form a sufficiently stable complex with p/t DNA (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). In contrast, pol V Mut E38K/ΔC17, which forms a much more stable complex with p/t DNA can synthesize DNA in the presence of either ATP or ATPγS (<xref ref-type="fig" rid="fig3">Figure 3F</xref>). To determine if pol V Mut WT can synthesize DNA in the presence of ATP (as must presumably happen in vivo) we have used the β sliding clamp to enhance pol V Mut binding to the 3′-primer end. Pol V Mut WT binds to β clamp and is able to synthesize DNA with moderately high processivity (<xref ref-type="bibr" rid="bib17">Karata et al., 2012</xref>). To determine if increased binding stability for pol V Mut WT might enable it to use ATP for synthesis, we used a p/t DNA with a 12 nt oh-containing streptavidin attached at the 5′-template end and the hairpin loop, preventing the β clamp from sliding off the DNA (<xref ref-type="bibr" rid="bib23">Schlacher et al., 2006</xref>). Pol V Mut WT in the presence of ATP (in the absence of ATPγS) incorporates 12 nt processively to reach the end of the template strand (<xref ref-type="fig" rid="fig5">Figure 5</xref>). The presence of ATP in the reaction, which is required to load β clamp (<xref ref-type="bibr" rid="bib1">Bertram et al., 2000</xref>), is able to support DNA synthesis. However, the addition of ATPγS significantly stimulates synthesis (<xref ref-type="fig" rid="fig5">Figure 5</xref>), presumably by further enhancing binding to p/t DNA (<xref ref-type="fig" rid="fig5">Figure 5</xref>; <xref ref-type="table" rid="tbl2">Table 2</xref>), and by reducing the unloading of β clamp by the γ complex (<xref ref-type="bibr" rid="bib1">Bertram et al., 2000</xref>).<fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.02384.017</object-id><label>Figure 5.</label><caption><title>Pol V Mut WT is active with ATP only in the presence of β/γ complex.</title><p>Sketch of the experimental set up is illustrated above the gels. To prevent the β clamp from sliding off the DNA, a 12 nt oh HP was designed containing biotin/streptavidin on both sides of the primer terminus substrate. The activity of pol V Mut WT was measured in the presence and absence of β/γ. In the presence of β/γ complex (left panel) pol V Mut WT is able to extend p/t with ATP, in contrast no DNA synthesis is observed with ATP in the absence of β/γ complex (right panel).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02384.017">http://dx.doi.org/10.7554/eLife.02384.017</ext-link></p></caption><graphic xlink:href="elife02384f005"/></fig></p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>Mutagenic DNA synthesis during the SOS response is an act of cellular desperation, and it comes with a price. However, when pol V Mut is activated and arrives on the scene, mutagenesis is not indiscriminate. Pol V Mut possesses a novel mechanism with which to limit processivity and restrict mutagenic DNA synthesis to those short DNA segments where it is required. In essence, the enzyme has evolved to do the absolute minimum to get cellular DNA synthesis restarted. No other DNA polymerase characterized to date has either an intrinsic ATPase activity or a similar autoregulatory mechanism.</p><p>We have previously shown that the active form of DNA polymerase V is UmuD′<sub>2</sub>C-RecA-ATP (<xref ref-type="bibr" rid="bib16">Jiang et al., 2009</xref>), but the roles of ATP and RecA in polymerase function were unknown. In vitro results using RecA variants that provide more facile activation and/or lack intrinsic ATPase function now elucidate the role of ATP. ATP or an ATP homologue is required for pol V Mut function (<xref ref-type="fig" rid="fig1">Figure 1B</xref>). Further, pol V Mut is a DNA-dependent ATPase (<xref ref-type="fig" rid="fig2">Figure 2A,B</xref>), which binds ATP in the absence of DNA (<xref ref-type="fig" rid="fig2">Figure 2C</xref>). Hydrolysis of ATP by pol V Mut results in dissociation of the enzyme from DNA (<xref ref-type="fig" rid="fig4">Figure 4</xref>). If ATPγS is used such that ATP is not hydrolyzed, then the enzyme remains stably bound to DNA (<xref ref-type="fig" rid="fig4">Figure 4</xref>, <xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2</xref>). This is the only DNA polymerase studied to date that is regulated by ATP binding and hydrolysis.</p><p>Activation of pol V to pol V Mut requires transfer of a RecA monomer from the 3′-proximal tip of RecA* (<xref ref-type="bibr" rid="bib23">Schlacher et al., 2006</xref>) (see e.g., <xref ref-type="fig" rid="fig1">Figure 1A</xref>). The ATP hydrolytic sites in RecA protein filament are situated at the interfaces of adjacent RecA subunits, and each neighboring subunit contributes key residues to the active site. There are no readily identifiable ATP-binding motifs present in either the UmuD′ or UmuC subunits of pol V. The avid ATPase activity observed with the ATPase-deficient RecA E38K/K72R indicates that the RecA subunit is not contributing a Walker A motif (or P-loop) to the aggregate site. We speculate that the ATPase active site in pol V Mut is newly created when RecA is added to the complex during activation, perhaps at the interface between the RecA subunit and UmuC or UmuD′. This interface appears to play a central role in polymerase activation because the RecA(S117F) mutant (initially referred to as RecA1730), was shown to be SOS-non mutable in vivo (<xref ref-type="bibr" rid="bib8">Dutreix et al., 1989</xref>). The S117 residue is situated at the 3′-proximal tip of RecA* (<xref ref-type="bibr" rid="bib26">Sommer et al., 1998</xref>), and, notably, pol V Mut S117F has no measurable DNA synthesis activity in vitro (<xref ref-type="bibr" rid="bib24">Schlacher et al., 2005</xref>).</p><p>In the cell pol V is post-transcriptionally regulated through proteolysis (<xref ref-type="bibr" rid="bib10">Frank et al., 1993</xref>; <xref ref-type="bibr" rid="bib12">Gonzalez et al., 1998</xref>, <xref ref-type="bibr" rid="bib13">2000</xref>) and by RecA*(<xref ref-type="bibr" rid="bib4">Burckhardt et al., 1988</xref>; <xref ref-type="bibr" rid="bib21">Nohmi et al., 1988</xref>; <xref ref-type="bibr" rid="bib25">Shinagawa et al., 1988</xref>; <xref ref-type="bibr" rid="bib23">Schlacher et al., 2006</xref>), presumably to ensure that this low fidelity pol (<xref ref-type="bibr" rid="bib22">Reuven et al., 1999</xref>; <xref ref-type="bibr" rid="bib29">Tang et al., 1999</xref>) is used only in dire circumstances. When the regulation of pol V fails, as it does for the constitutive RecA E38K mutant, pol V Mut is induced in the absence of DNA damage generating ∼100-fold increase in mutations (<xref ref-type="bibr" rid="bib30">Witkin, 1967</xref>). This huge increase in mutations likely occurs by copying undamaged DNA with exceptionally poor fidelity (<xref ref-type="bibr" rid="bib28">Tang et al., 2000</xref>). The regulation of pol V is needed to limit mutations, especially in stationary phase cells (<xref ref-type="bibr" rid="bib6">Corzett et al., 2013</xref>). Our biochemical data suggest that in vivo ATP adds another level to this regulation; not only is the timing of pol V activity regulated, but also its access to p/t DNA. A model for the role of ATP in pol V Mut activity is sketched in <xref ref-type="fig" rid="fig6">Figure 6</xref>. ATP is required to bind pol V Mut to DNA. ATP hydrolysis releases the enzyme from DNA, which in vivo would ensure that tracts of DNA synthesized by pol V Mut are short, limiting the opportunity for misincorporation to the region immediately adjacent to the template lesion. Therefore, the internally regulated DNA-dependent ATPase of pol V Mut provides a way to limit mutational load.<fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.02384.018</object-id><label>Figure 6.</label><caption><title>Model showing ATP regulation of pol V Mut activity.</title><p>Pol V Mut is active for DNA synthesis only after binding a molecule of ATP (green triangle) to form UmuD′<sub>2</sub>C-RecA-ATP. The binding of ATP is required for polymerase association with p/t DNA. ATP-hydrolysis catalyzed by an intrinsic DNA-dependent ATPase triggers pol V Mut-p/t DNA dissociation, while leaving intact the UmuD′<sub>2</sub>C-RecA complex.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02384.018">http://dx.doi.org/10.7554/eLife.02384.018</ext-link></p></caption><graphic xlink:href="elife02384f006"/></fig></p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>DNA oligos</title><p>DNA oligos were synthesized using a 3400 DNA synthesizer (Applied Biosystems/Life Technologies, Carlsbad, CA). Oligo modifications (Flourescein-dT Phosphoramidite, Biotin-dT, and 5′-Amino-Modifier C12) were purchased from Glen Research. DNA sequences are in <xref ref-type="table" rid="tbl3">Table 3</xref>.<table-wrap id="tbl3" position="float"><object-id pub-id-type="doi">10.7554/eLife.02384.019</object-id><label>Table 3.</label><caption><p>Sequences for p/t HP DNA</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02384.019">http://dx.doi.org/10.7554/eLife.02384.019</ext-link></p></caption><table frame="hsides" rules="groups"><tbody><tr><td colspan="2">Sequences for p/t HP DNA</td></tr><tr><td> 3 nt oh HP</td><td>5′ AGA GCA GTT AGC GCA TTC AGC TCA TAC TGC TGA ATG CGC TAA CTG C 3′</td></tr><tr><td> 3 nt oh HP (TTT)</td><td>5′ TTT GCA GTT AGC GCA TTC AGC TCA TAC TGC TGA ATG CGC TAA CTG C 3′</td></tr><tr><td> Fluorescein (<bold>FAM</bold>) 3 nt oh HP</td><td>5′ AGA GCA GTT AGC GCA T(<bold>FAM</bold>)C AGC TCA TAC TGC TGA ATG CGC TAA CTG C 3′</td></tr><tr><td> 12 nt oh HP</td><td>5′ CGA AAC AGG AAA GCA GTT AGC GCA TTC AGC TCA TAC TGC TGA ATG CGC TAA CTG C 3′</td></tr><tr><td> Fluorescein (<bold>FAM</bold>) 12 nt oh HP</td><td>5′ CGA AAC AGG AAA GCA GTT AGC GCA TTC AGC TCA TAC TGC TGA A(<bold>FAM</bold>)G CGC TAA CTG C 3′</td></tr><tr><td> Biotinylated (<bold>Bio</bold>) 12 nt oh HP</td><td>5′ (<bold>Bio</bold>)GA AAC AGG AAA GCA GTT AGC GCA TTC AGC (<bold>Bio</bold>)CA TAC TGC TGA ATG CGC TAA CTG C 3′</td></tr><tr><td colspan="2"><bold>Sequences for pol V Mut assembly, activity and ATP hydrolysis DNA</bold></td></tr><tr><td> Amino C12-linked 45mer attached to cyanogen bromide-activated sepharose resin</td><td>5′ <bold>C12</bold>TT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT T 3′</td></tr><tr><td> ssDNA 30 mer for RecA transactivation and ATP hydrolysis</td><td>5′ ACT GAC CCC GTT AAA ACT TAT TAC CAG TAA 3′</td></tr></tbody></table></table-wrap></p></sec><sec id="s4-2"><title>Proteins</title><p>His-tagged pol V was purified from <italic>E. coli</italic> stain RW644 as described by <xref ref-type="bibr" rid="bib17">Karata et al. (2012)</xref> and RecA WT was purified by a standard protocol (<xref ref-type="bibr" rid="bib7">Cox et al., 1981</xref>). RecA E38K/K72R and RecA E38K/ΔC17 were provided by Michael Cox at the University of Wisconsin, Madison. We incorporated <italic>p</italic>-azido-L-phenylalanine (<italic>p</italic>AzF) (<xref ref-type="bibr" rid="bib5">Chin et al., 2002</xref>) into RecA WT to site specifically label the protein with Alexa Fluor 488 DIBO alkyne. For the cloning of RecA, we used the pAIR79 plasmid, which was a gift from Michael Cox at the University of Wisconsin, Madison. The Phe21 sequence in RecA WT was replaced with the amber codon via site-directed mutagenesis using Pfu Ultra polymerase (Agilent Technologies). Once the sequence was confirmed, pAIR79 was cotransformed with the vector pEVOL-<italic>p</italic>AZF (a gift from the Peter Schultz lab at The Scripps Research Institute, San Diego, CA) into the BLR expression strains (<xref ref-type="bibr" rid="bib32">Young et al., 2010</xref>). The RecA<sub>F21AzF</sub> protein was purified using the same standard protocol for RecA WT (<xref ref-type="bibr" rid="bib7">Cox et al., 1981</xref>). RecA<sub>F21AzF</sub> was labeled with Alexa Flour 488 (RecA<sub>F21AzF</sub>-Alexa Fluor 488) according to manufacturer's instructions (Life Technologies).</p></sec><sec id="s4-3"><title>Pol V Mut assembly</title><p>5′ amino-modified 45 nt ssDNA was covalently attached to Cyanogen-Bromide Sepharose resin according to manufacturer's protocol (Sigma–Aldrich) and transferred to a spin column (Biorad). Briefly, the resin was activated with 1 mM HCl then washed with water and equilibrated with coupling buffer (0.1 M NaHCO<sub>3</sub>, 0.5 M NaCl). 5′amino-modified DNA (20 nmoles) was incubated with 100 mg resin overnight at 4°C, and unbound oligomers were removed by washing resin seven times with coupling buffer. Reactive amino groups were blocked with Ethanolamine (1 M, pH 8; Sigma–Aldrich) to prevent non-specific binding then stored in 1 M NaCl. The concentration of 45 nt ssDNA bound to the resin consistently provided about 6 nmoles per 100 mg of resin. Stable RecA* was assembled by incubating excess RecA or RecA mutants and ATPγS (Roche) or ATP (Amersham-Pharmacia) when stated with 0.5 nmole ssDNA-bound resin for 20 min at 37°C. Free RecA and ATPγS were separated from RecA*-resin by gentle centrifugation at 0.1×<italic>g</italic> for 1 min and collected in the flow through. Washes were repeated with reaction buffer (20 mM Tris–HCl pH 7.5, 25 mM Sodium Glutamate, 8 mM MgCl<sub>2</sub>, 8 mM DTT, 4% glycerol, 0.1 mM EDTA) until no RecA was detected in the flow through. His-tagged pol V (2 nmole) was resuspended in reaction buffer and mixed with RecA*-resin to form pol V Mut. The pol V-RecA*-resin suspension was incubated at 37°C for 15 min followed by centrifugation (0.1×<italic>g</italic> for 1 min) to separate pol V Mut from the RecA*-resin in the spin column. The concentration of pol V Mut collected in the flow through was determined by SDS-PAGE gel. Briefly, pol V and RecA proteins at various known concentrations were resolved on an SDS-PAGE gel (10%) as standards and gel band intensities were quantified using IMAGEQUANT software. Standard gel intensities of UmuC and RecA were then used to determine the concentration of pol V Mut.</p></sec><sec id="s4-4"><title>DNA extension</title><p>The activity of pol V Mut was detected via DNA extension on a 5′-<sup>32</sup>P-labeled primer template hairpin with a 3-nt overhang. Pol V Mut (400 nM) was added to a 10-μl reaction mixture containing annealed template DNA (50 nM), ATP, ATPγS, dATP, GTP, dTTP, or ADP (500 μM, unless stated otherwise) and dNTPs (Amersham-Pharmacia) (500 μM each). Substrate DNA was preincubated with first streptavidin (400 nM) then β/γ complex (250 nM and 100 nM respectively) (a gift from Linda Bloom at the University of Florida, Gainesville) when present. Reactions were carried out at 37°C for 30 min. To detect free RecA in the pol V Mut solution, ssDNA (50 nM) was added to the reaction and activity was measured in the presence of ATPγS. Comparable activity levels between ATPγS alone and ATPγS + ssDNA indicate that pol V Mut is intact and free of RecA that is not part of the mutasome. Reactions were resolved on a 20% denaturing polyacrylamide gel allowing single nucleotide separation. Gel band intensities were detected by phosphorimaging and quantified using IMAGEQUANT software. Primer utilization was calculated as the unextended primer intensity subtracted from the total DNA intensity giving the percentage of primer utilized.</p></sec><sec id="s4-5"><title>Pol V Mut and RecA-dependent P<sub>i</sub> release (ATP hydrolysis)</title><p><italic>E. coli</italic> phosphate-binding protein (PBP) was purified and labeled with MDCC fluorophore (Life Technologies) according to the protocol from <xref ref-type="bibr" rid="bib3">Brune et al. (1998)</xref>. Binding of P<sub>i</sub> (phosphate) by MDCC-PBP is rapid and tight (Kd ∼ 0.1 µM) resulting in a large increase in fluorescence (<xref ref-type="bibr" rid="bib3">Brune et al., 1998</xref>). The change in fluorescence of MDCC-PBP was detected in real-time using a QuantaMaster (QM-1) fluorometer (Photon Technology International). Wavelengths for excitation and emission of the MDCC were selected using monochromators with a 1-nm band pass width. Excitation and emission were set at 425 and 464 nm, respectively. A 65-μl aliquot of 5 μM MDCC-PBP was incubated with 0.05 units/ml PNPase, 100 μM 7-methylguanosine and various concentrations of pol V Mut or RecA. The PNPase and 7-methylguanosine were used to remove any traces of Pi in the reaction prior ATP hydrolysis. The time-based scan was initiated for about 1000 s. ATP hydrolysis was initiated by adding a 5-μl mixture of ATP and DNA to final concentrations of 500 μM and 1 μM, respectively and the measurements were taken at 1 point per sec resolution. The maximum rate (<italic>V</italic><sub>max</sub>) of P<sub>i</sub> release was derived from the linear slope of Pi release, and <italic>k</italic><sub>cat</sub> was calculated by dividing <italic>V</italic><sub>max</sub> by the enzyme concentration. Each measurement was repeated 2–3 times.</p></sec><sec id="s4-6"><title>Steady-state rotational anisotropy binding assay and pol V Mut off-rate</title><p>Pol V Mut binding to p/t DNA was measured by changes in steady-state fluorescence depolarization (rotational anisotropy). Reactions (70 μl) were carried out at 37°C in standard reaction buffer, and contained a fluorescein-labeled hairpin DNA (50 nM), 500 μM ATP or ATPγS and varied concentrations of pol V Mut. For ATP titration experiments, fluorescein-labeled hairpin DNA (50 nM) and pol V Mut were mixed together in reaction buffer and ATP or ATPγS was titered to a final concentration of 750 μM. Rotational anisotropy was measured using a QuantaMaster (QM-1) fluorometer (Photon Technology International) with a single emission channel. Samples were excited with vertically polarized light at 495 nm and both vertical and horizontal emission was monitored at 520 nm.</p><p>To measure the enzyme's off-rate, first pol V Mut E38K/ΔC17 was prebound to 50 nM fluorescein-labeled hairpin DNA with a 12 nt single-stranded overhang in the presence of 500 μM ATP or 500 μM ATPγS. The enzyme off-rate was measured by monitoring changes in rotational anisotropy after the addition of excess trap, unlabeled DNA (8 μM). The off-rate was fit to single exponential decay. Anisotropy for free DNA and pol V Mut E38K/ΔC17 in the absence of trap was measured in the same experiment.</p><p>To determine the integrity of pol V Mut during ATP hydrolysis and DNA synthesis we employed the use of site-specifically labeled RecA<sub>F21AzF</sub>-Alexa Fluor 488 to assemble pol V Mut WT<sub>F21AzF</sub>-Alexa Fluor 488. Pol V Mut WT<sub>F21AzF</sub>-Alexa Fluor 488 (100 nM) was mixed with dNTPs (500 μM) to a final volume of 70 μl and the rotational anisotropy was measured. To the same cuvette, ATP or ATPγS (500 μM) was added for another measurement. To create DNA synthesis conditions, 12 nt oh HP (1 μM) was added to the cuvette and rotational anisotropy was measured at 2 min, 5 min, and 10 min. The rotational anisotropy of RecA<sub>F21AzF</sub>-Alexa Fluor 488 (100 nM) was measured in parallel to pol V Mut WT<sub>F21AzF</sub>-Alexa Fluor 488.</p></sec><sec id="s4-7"><title>Binding of pol V Mut to etheno-ATP</title><p>Pol V Mut binding to etheno-ATP (Life Technologies) was measured as a change in rotational anisotropy at three different etheno-ATP concentrations, 500 μM, 750 μM, and 1000 μM. In a 70-μl reaction, etheno-ATP was mixed in standard reaction buffer with pol V Mut, pol V or RecA. The concentration of protein used for measurements was 400 nM. Rotational anisotropy was measured using a QuantaMaster (QM-1) fluorometer. Samples were excited with vertically polarized light at 410 nm and both vertical and horizontal emissions were monitored at 425 nm.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>ALE and MJ contributed to this work equally. We thank Dr Linda B Bloom, U Florida, Gainesville, for a generous gift of purified β/γ complex proteins and Dr Peter G Schultz, Scripps La Jolla, for kindly providing us with the vector pEVOL-<italic>p</italic>AZF.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>ALE, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>MJ, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>JGB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>RW, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con5"><p>MFG, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con6"><p>MMC, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn></fn-group></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bertram</surname><given-names>JG</given-names></name><name><surname>Bloom</surname><given-names>LB</given-names></name><name><surname>Hingorani</surname><given-names>MM</given-names></name><name><surname>Beechem</surname><given-names>JM</given-names></name><name><surname>O'donnell</surname><given-names>M</given-names></name><name><surname>Goodman</surname><given-names>MF</given-names></name></person-group><year>2000</year><article-title>Molecular mechanism and energetics of clamp assembly in <italic>Escherichia coli</italic>. 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id="SA1"><front-stub><article-id pub-id-type="doi">10.7554/eLife.02384.020</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Botchan</surname><given-names>Michael R</given-names></name><role>Reviewing editor</role><aff><institution>University of California, Berkeley</institution>, <country>United States</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “DNA polymerase V activity is autoregulated by a novel intrinsic DNA-dependent ATPase” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by a Senior editor and 3 reviewers, one of whom is a member of our Board of Reviewing Editors.</p><p>The following individuals responsible for the peer review of your submission have agreed to reveal their identity: Michael Botchan (Reviewing editor) and Michael O'Donnell (peer reviewer).</p><p>The Reviewing editor and the other reviewers discussed their comments before reaching this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>This is an important study, providing interesting answers to questions raised by previous work from the collaborating authors’ labs; why does the enzyme require ATP and what does the ribonucleotide do for the synthesis. The data clearly show that the SOS activated DNA Pol V, containing a single RecA molecule as part of the holoenzyme, requires ATP binding for association with a primer/template substrate and that hydrolysis leads to dissociation. The results are clearly presented and the data do provide very strong support for the idea that ATP hydrolysis serves as a switch to prevent the mutation prone polymerase from staying on the chromosome for too long and creating too many unwarranted mutations. It leaves the readers with the thought that during evolution, the Pol V co-opted the RecA, piggy-backing on it to use the RecA ATPase properties for its (Pol V) own design. Thus, RecA binds ATP in order to bind DNA, and hydrolyzes ATP to eject from DNA – and Pol V does the same. It is really quite brilliant and makes an excellent story about evolution of an enzyme that adapts to obtain extended mechanical features over those of simpler polymerases. The story should be published in <italic>eLife</italic>. A couple of points should be addressed in a simple revision.</p><p>1) The work relies heavily on mutant forms of RecA, particularly a mutant in the Walker A domain of the protein. The activated Pol V assembled with the mutant is thought to have an interesting hybrid ATP hydrolysis site where one or another surface of the other proteins may provide a residue compensating for the loss of the charge in the RecA. Two related views on this point arise: this mutant situation works well for synthesis and actually better than wt.</p><p>a) Can one eliminate the thought that in the wt the substitute surface actually doesn't come into play or do the authors believe that allostery distorts the well-studied RecA Walker A region? This point speaks to how “distinct” the RecA activity truly is in the polymerase or casts a shadow on where the ATPase site really lies.</p><p>b) Considering that the ATPase properties of RecA as a monomer may not have been studied before, it is conceivable that the conservative K to R replacement becomes functional in the context of a monomeric RecA. Though not essential we suggest the following – either for this paper or for a discussion of future directions: reconstitute pol V Mut with a RecA mutant that can no longer bind ATP (e.g., less conservative mutant) and determine if it still binds etheno-ATP, and to use the same mutant in ATPase assays. It seems easy to try, and may help support the interesting evolutionary scenario of a polymerase capturing another enzyme and harnessing its ATP-DNA cycle for enhanced mechanical function.</p><p>2) The wt activated Pol V actually hydrolyses ATP so well that essentially no synthesis is detected unless a non-hydrolyzable nucleotide is in the mix. With the mutant RecA enzymes ATPγS actually stimulates synthesis. It would be reassuring to show that for the wt and with quantification, changing the ratios of ATPγ to ATP leads to anticipated changes in synthesis. Quantification here might help with the next point.</p><p>3) There is little indication of the reproducibility of the experiments presented. Not one data plot in any of the figures have error bars nor do some of the tables give standard deviations. We would like to see this fixed in the revisions – one assumes that quantification of gel results with numbers of fold differences might be possible in some key experiments or with plots as indicated above.</p><p>4) For directly testing the switch and release idea – would using a much longer tail allow for a measurement of lengths actually synthesized? We anticipate that with a very long template only very short oligos would be made with ATP but at higher levels of the analogue longer lengths would be measured.</p><p>5) The authors suggest that pol V Mut remains intact in the presence of ATP/ATPγS, even during DNA synthesis. This conclusion needs to be firmed up. The experiment presented (<xref ref-type="fig" rid="fig4s2">Figure 4–figure supplement 2</xref>) uses the 12 oh p/t and the rotational anisotropy remains constant for up to 10 minutes. But what are the kinetics of DNA synthesis? They are not presented on either the 3 oh or 12 oh template, only 30 minute incubations are shown. So, is there DNA synthesis occurring under the conditions and time period of the rotational anisotropy experiment?</p><p>6) The extent of DNA synthesis shown in <xref ref-type="fig" rid="fig5">Figure 5</xref> seems very low. The figure is over-exposed (for the starting material) that one cannot determine whether the first one or two nucleotides are being polymerized. It is difficult to compare this figure with, say, <xref ref-type="fig" rid="fig1">Figure 1</xref>, where extension of the first nucleotide seems quite efficient. Similarly, the extent of DNA synthesis seems to be less than previously reported in <xref ref-type="fig" rid="fig5">Figure 5</xref> of Karata et al. (referenced within). What is the extent of synthesis?</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.02384.021</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) The work relies heavily on mutant forms of RecA, particularly a mutant in the Walker A domain of the protein. The activated Pol V assembled with the mutant is thought to have an interesting hybrid ATP hydrolysis site where one or another surface of the other proteins may provide a residue compensating for the loss of the charge in the RecA. Two related views on this point arise: this mutant situation works well for synthesis and actually better than wt</italic>.</p><p><italic>a) Can one eliminate the thought that in the wt the substitute surface actually doesn't come into play or do the authors believe that allostery distorts the well-studied RecA Walker A region? This point speaks to how “distinct” the RecA activity truly is in the polymerase or casts a shadow on where the ATPase site really lies</italic>.</p><p>RecA* nucleoprotein filament formed with the “non-ATP hydrolyzable” RecA E38K/K72R mutant has no detectable DNA-dependent ATP hydrolysis activity at RecA concentrations (0.2 and 0.4 μM). The same concentrations with pol V Mut E38K/K72R show significant DNA-dependent ATP hydrolysis, at rates that increase with increasing protein concentration (<xref ref-type="fig" rid="fig2">Figure 2B</xref>). The weak DNA-dependent ATP hydrolysis detected at 2.5 μM RecA E38K/K72R (<xref ref-type="fig" rid="fig2s1">Figure 2–figure supplement 1</xref>), is less than the rate of hydrolysis observed using a 25-fold lower concentration of pol V Mut E38K/K72R (0.1 μM, <xref ref-type="fig" rid="fig2">Figure 2B</xref>).</p><p>The strong DNA-dependent ATP hydrolysis observed when RecA E38K/K72R is used to form pol V Mut suggests that a distinct ATP hydrolysis pocket is formed. The much higher rate of ATP hydrolysis for pol V Mut WT (<xref ref-type="fig" rid="fig2">Figure 2A</xref>) compared to pol V Mut E38K/K72R (<xref ref-type="fig" rid="fig2">Figure. 2B</xref>) suggests that the K72 residue in RecA, while not crucial to hydrolysis in pol V Mut, may well be proximal to the ATPase active site, or at least close enough to cause a significant reduction in the hydrolysis rate. The K72 residue (changed to R in the mutant) is at the 3′-proximal end of the RecA filament, and thus one might expect this surface to be close to UmuD′<sub>2</sub>C in the activated complex. At present, we can only say that the ATPase active site in pol V Mut does not feature the RecA K72 residue in its normal role.</p><p><italic>b) Considering that the ATPase properties of RecA as a monomer may not have been studied before, it is conceivable that the conservative K to R replacement becomes functional in the context of a monomeric RecA. Though not essential we suggest the following – either for this paper or for a discussion of future directions: reconstitute pol V Mut with a RecA mutant that can no longer bind ATP (e.g., less conservative mutant) and determine if it still binds etheno-ATP, and to use the same mutant in ATPase assays. It seems easy to try, and may help support the interesting evolutionary scenario of a polymerase capturing another enzyme and harnessing its ATP-DNA cycle for enhanced mechanical function</italic>.</p><p>It is not possible to make pol V Mut with RecA that does not bind ATP. A mutant unable to bind ATP will not form a RecA* filament, and the assembly of an active pol V Mut requires pol V to interact with the 3′-tip of the RecA* nucleoprotein filament, which is required to transfer a RecA monomer to UmuD′<sub>2</sub>C (Jiang et al. 2009. The active form of DNA polymerase V is UmuD′<sub>2</sub>C-RecA-ATP. Nature, 460:359-63). We note that RecA cannot hydrolyze ATP as a monomer. In a RecA filament, the RecA active site for ATP hydrolysis is a composite site that is formed at the interface of two RecA subunits, both of which contribute residues essential to active site function. Thus, a RecA filament can hydrolyze ATP, a monomer cannot.</p><p><italic>2) The wt activated Pol V actually hydrolyses ATP so well that essentially no synthesis is detected unless a non-hydrolyzable nucleotide is in the mix. With the mutant RecA enzymes ATPγS actually stimulates synthesis. It would be reassuring to show that for the wt and with quantification, changing the ratios of ATPγ to ATP leads to anticipated changes in synthesis. Quantification here might help with the next point</italic>.</p><p>We have performed the experiment suggested by the referees by changing the ratio of ATPγS/ATP, and obtained the anticipated result that DNA synthesis measured with ATPγS is reduced significantly in the presence of ATP. We measured DNA synthesis with pol V Mut WT as a function of ATPγS concentration, in the presence and absence of ATP. ATP (500 μM) reduces primer elongation (<xref ref-type="fig" rid="fig3s2">Figure 3–figure supplement 2</xref>, revised manuscript), which suggests that the fraction of enzyme that binds and hydrolyzes ATP is not associated with DNA long enough to observe activity.</p><p><italic>3) There is little indication of the reproducibility of the experiments presented. Not one data plot in any of the figures have error bars nor do some of the tables give standard deviations. We would like to see this fixed in the revisions – one assumes that quantification of gel results with numbers of fold differences might be possible in some key experiments or with plots as indicated above</italic>.</p><p>Error bars have been added to figures and standard errors of the mean have been included in the tables.</p><p><italic>4) For directly testing the switch and release idea – would using a much longer tail allow for a measurement of lengths actually synthesized? We anticipate that with a very long template only very short oligos would be made with ATP but at higher levels of the analogue longer lengths would be measured</italic>.</p><p>We performed the suggested experiment, which is included as <xref ref-type="fig" rid="fig4s1">Figure 4–figure supplement 1</xref> in the revised manuscript. We measured DNA synthesis on a 12 nt oh HP with pol V Mut E38K/ ΔC17 as a function of ATP and ATPγS concentration. As expected short segments of DNA are made with ATP and much longer segments are synthesized with ATPγS, and primer extension lengths are greater at higher ATPγS concentrations.</p><p><italic>5) The authors suggest that pol V Mut remains intact in the presence of ATP/ATPγS, even during DNA synthesis. This conclusion needs to be firmed up. The experiment presented (<xref ref-type="fig" rid="fig4s2">Figure 4–figure supplement 2</xref>) uses the 12 oh p/t and the rotational anisotropy remains constant for up to 10 minutes. But what are the kinetics of DNA synthesis? They are not presented on either the 3 oh or 12 oh template, only 30 minute incubations are shown. So, is there DNA synthesis occurring under the conditions and time period</italic> <italic>of the rotational anisotropy experiment?</italic></p><p>We have measured the DNA synthesis rates under the conditions (100 nM pol V Mut and 1 μM HP) and time period of the rotational anisotropy experiment; the data are shown in <xref ref-type="fig" rid="fig4s3">Figure 4–figure supplement 3B</xref>, in the revised manuscript. Pol V Mut activity data were obtained at 2, 5, 10, 20 and 30 min, and synthesis is observed at 5, 10 and 30 min (at this high (10:1) DNA/pol V Mut ratio used to measure rotational anisotropy of RecA bound to UmuD′<sub>2</sub>C in the pol V Mut complex, primer extension is below the level of detection at 2 min). Therefore, we observe DNA synthesis occurring under the conditions and time period of the rotational anisotropy measurement for pol V Mut RecA<sub>F21AzF</sub> -Alexa Fluor 488, similar to synthesis observed with the non-fluorescently labeled pol V Mut RecA WT (<xref ref-type="fig" rid="fig4s3">Figure 4–figure supplement 3B</xref>).</p><p><italic>6) The extent of DNA synthesis shown in <xref ref-type="fig" rid="fig5">Figure 5</xref> seems very low. The figure is over-exposed (for the starting material) that one cannot determine whether the first one or two nucleotides are being polymerized. It is difficult to compare this figure with, say, <xref ref-type="fig" rid="fig1">Figure 1</xref>, where extension of the first nucleotide seems quite efficient. Similarly, the extent of DNA synthesis seems to be less than previously reported in <xref ref-type="fig" rid="fig5">Figure 5</xref> of Karata et al. (referenced within). What is the</italic> <italic>extent of synthesis?</italic></p><p>In the Karata et al. reference, a different DNA substrate was used (circular p/t DNA) that could account for an increased rate of DNA synthesis. We also suspect that there had been a small amount of adventitious RecA present in the earlier report that could assemble as RecA* on unannealed primer strands and increase synthesis by transactivation of pol V Mut. Our significantly improved method of assembling pol V Mut using ssDNA covalently attached to Sepharose beads (<xref ref-type="fig" rid="fig1">Figure 1A</xref>) eliminates ATP/ATPγS (and free RecA). The purpose of this experiment (<xref ref-type="fig" rid="fig5">Figure 5</xref>) was to determine if activity for pol V Mut WT can be obtained with ATP in the presence of the β clamp. The overall activity in the presence of ATP is low as a result of the dynamic behavior of γ complex using ATP to load and unload β. Additionally in these experiments the HP p/t DNA has biotin/streptavidin to prevent the loaded β clamp from sliding off, which partially inhibits pol V Mut activity. The key point is that pol V Mut WT DNA synthesis occurs with ATP in the presence of β/γ complex.</p></body></sub-article></article>