2558 Cell Cycle 12:16,2558–2563;August 15,2013;©2013LandesBioscience http://dx.doi.org/10.4161/cc.25691 07/09/2013 Accepted: 06/24/2013 Submitted: of ALT, lengthening tion; alternative replica- break-induced BIR, ment loop; pol, DNAD-loop, ; - displace break; double-strand DSB, replication; directed recombination RDR, bination; Email: [email protected] *Correspondence to: Richard T Pomerantz; Abbreviations: replication break-induced esis, mutagen adaptive , stress-induced repair, break strand double- recombination, homologous DNA replication, Keywords: 1 Pomerantz T Richard intermediates recombination error-proneDNA are at RecA Sciences; University of Southern California; Los Angeles, CA USA; USA; CA Angeles, Los California; ofSouthern University Sciences; Fels Institute for Cancer Research; Department of Biochemistry; Temple University School of Medicine; Philadelphia, PA USA; PA USA; Philadelphia, ofMedicine; School Temple University ofBiochemistry; Department Research; Cancer for Institute Fels

P ap er ty p e HR, homologous recom homologous HR, 1, *, Myron FGoodman - - G 2 now evident that stress-induced mutations stress-induced that now evident major threat to modern medicine. to modern major threat a becoming is which resistance, antibiotic to promote shown been has mutagenesis sure to antibiotics. to sure expo and nutrient starvation as such tions, condi environmental adverse overcome to bacteria allows that process evolutionary amajor is mutagenesis, stress-induced proliferating cells. non-stressed in RDR during mutations for suppressing necessary are factors auxiliary that suggest and D-loops error-prone RecA-mediated at sically intrin are DNA polymerases that cate indi IV. findings pol These like D-loops error-prone at highly is DNA synthesis, high-fidelity exhibits normally which pol I, A-family that finding surprising the demonstrate View,we Extra this In D-loops. at mutagenesis stress-induced promoting in IV pol implicated have that data genetic longstanding verify findings These vitro. in RDR during mutagenic highly is and concentrations induced at stress- intermediates recombination D-loop to recruited preferentially is IV pol that demonstrated have we studies recent In cells. coli E. growth-limited in repair (DSB) break double-strand ing dur promote to mutations ability its to due of stress conditions under (RDR) replication recombination-directed error-prone performs (DinB) IV merase , and Michael EO’Donnell Michael , and Adaptive mutagenesis, also known as as known also mutagenesis, Adaptive Y-family translesion DNA poly translesion Y-family that suggested have studies enetic 3 The Rockefeller University; Howard Hughes Medical Institute; New York, NY USA York, NY New Institute; Medical Hughes Howard University; Rockefeller The Introduction C ell Cycle 1-3 For example, adaptive adaptive For example,

3 ‑ m 4,5 ediated ediated It is ------under conditions of stress. conditions under of bacteria isolates natural and strains tory of labora promote survival DNA and the chromosomal and episomal on both occur limiting conditions limiting growth- under mutations (~85%) adaptive it promotes most that fore not surprising it there is DNA polymerase, Y-family error-prone an is IV pol that Considering cells. growth-limited in DNA polymerase abundant most the by far IV pol makes to develop resistance to therapy. resistance to develop cells of tumor ability the and progression for cancer implications have may therefore and stress and conditions growth-limiting to cells of cancer adaptation to the allels par many has mutagenesis stress-induced induced mutations. induced of stress- major driver the as DinB, as known also IV), (pol IV DNA polymerase translesion identified have Y-family which studies genetic of elegant years by several coli E. in mutagenesis (D-loop) recombination intermediates, intermediates, recombination (D-loop) loop displacement at or near mutations promotes adaptive IV pol that indicate the RpoS general stress response. stress general RpoS the by phase stationary during molecules/cell ~2-fold additional to ~5000 by an induced further is and response SOS the during to ~2500upregulated molecules/cell near DSBs. near to regions targeted mostly are mutations such and RuvABC, and RecBCD RecA, as such proteins DSB repair to require shown been have mutations adaptive induced pol IV- For example, studies. by genetic revealed been also has stress during activity The central mechanism of adaptive of adaptive mechanism central The Where pol IV elicits its mutagenic mutagenic its elicits IV pol Where 10 Hence, previous genetics genetics previous Hence, 2 Department of Biological ofBiological Department 8 1,2,8 . has been revealed revealed been has Pol IV is highly highly Pol is IV V 6,7 olume 12 Issue16 Importantly, Importantly, 3 8,9 This This - - -

©2013 Landes Bioscience. Do not distribute. www.landesbioscience.com yeast. in error-prone also is which (BIR), lication rep or break-induced extension D-loop as to referred often is process (RDR) cation repli recombination-directed This DSBs. to proximity close in mutations in ing result molecule, donor DNA undamaged the from information sequence the copies and co-factors, replication with along ably - D-loop,presum to the recruited then is aD-loop. in Pol donor IV duplex, resulting a homologous within invasion strand then promote filaments RecA DNA tails. 3 resulting the along tion forma filament promotes RecA DNA and the resects that complex nuclease/ RecBCD the by processed first are DSBs Figure in illustrated is DSB repair during mutagenesis stress-induced in involvement of polIV model general A for DSB repair. necessary are which prone RDR (stress-induced mutagenesis) mutagenesis) (stress-induced prone RDR DSB regions. DSB error-prone and SOS the by upregulation toits due cells growth-limited in to D-loops recruited is preferentially IV Pol formation. D-loop in resulting donor DNA ahomologous within invasion strand motes of loading facilitates and DNA the resects which by the processed DSBs are recombination. prone Figure 1. R e cA onto ssDNA. ssDNA. onto cA

P ap er ty R 11 Model of pol IV activity during error- during activity IV pol of Model ecBCD helicase-nuclease complex, Pol involvement error- in IV p R e D R R activity targets mutations to mutations targets activity p oS stress responses. Pol IV IV Pol responses. oS stress T h e

R e cA filament pro filament cA single-strand ′ single-strand

- 1 - - - - .

replication of such adducts. of such replication in of more pol IV,ortholog efficient also is to UV light. to UV by exposure caused frequently are which dimmers, pyrimidine cyclobutane as such due to damage DNA structures aberrant to accommodate them allows which sites, relativelypolymerases possess open active DNA low-fidelity Y-family polymerases, DNA to high-fidelity contrast In forks. replication do not arrest strand lagging the in lesions strand; leading the in lesions DNA with upon collision collapse or even arrested become forks replication tures, site struc active stringent due to their DNA in lesions upon encountering stall polymerases DNA replicative high-fidelity and oxidation, respectively. of hydrolysis result as environments lular cel unstressed normal in regularly occur which (8-oxoG), 7,8-dihydroguanine 8-oxo- and sites abasic include merases DNA poly by translesion bypassed lesions their respective lesions. proficientwhen and bypassing accurate relatively are they DNA, on undamaged error-prone mostly inefficient are and ases DNA polymer Y-family although that cates translesion synthesis (TLS). called a pathway in DNA lesions past tion to promote replica ability for their known typically are DNA polymerases Y-family to point out that it important is RDR, in . for this amajor activity likely is involvement RDR in IV pol nature, in conditions growth-limiting in periods spend extended bacteria Considering esis. mutagen of adaptive mechanism central the is cells growth-limited in activity RDR mutagenic pol IV that evidence compelling provided have studies genetic tionary-phase cells ( cells tionary-phase sta in occurs which response, stress RpoS of the induction and response SOS by the polymerase of the upregulation requires nine nucleotide pool, such as following following as pool, such nucleotide nine gua of the of oxidation aresult as occurs 8-oxo-dGTP, which to incorporate shown aged nucleotides. undam with compared N2-furfuryl-dG) N2-position (i.e., atthe adducts taining deoxyguanosine-con opposite replication in moreaccurate and is proficient IV pol Interestingly, mounting evidence indi Although pol IV is strongly implicated implicated strongly is IV pol Although Intriguingly, pol IV has also been been also has IV pol Intriguingly, 13-16 C 17 ell Cycle Other common DNA common Other Pol κ Pol Fig.

, the mammalian mammalian , the 14 1 ). For example, example, For 17 9,12 Altogether, Altogether, 13,14 Since Since ------for RDR. βfor not require does polymerase the that we found cells, SOS-induced in to those relative IV pol of concentrations Using activity. RDR IV for pol needed is onto DNA polymerases, the whether we examined response, SOS the during upregulated highly is IV pol Since DNA. on supercoiled activity placement dis strand robust exhibits and D-loops mediated to RecA recruited fore readily there Pol is conditions. IV identical under activity this to perform unable is V pol Y-family related the whereas RDR, proficientin is IV pol show that studies 8-oxo-dGMP lesions. 8-oxo-dGMP spaced of closely by repair incomplete ofcaused DSBs frequency due to ahigh presumably treatment, antibiotic during death cell to potentiate appears IV pol E. coli E. of stationary-phase studies genetic by previous suggested as promote RDR, IV to pol of ability the verify findings for β need precludethe may cells stressed in of IV pol levels abundant ases (I–V). Pol IV, II, involved (I–V). Vare and ases ity. activ RDR for by polIV accounted be may which stress, during replication in engaged actively is IV pol that indicates treatment antibiotic during incorporation of 8-oxo-dGTP frequency high suggested the elucidated, to be yet has 8-oxo-dGTP incorporates IV pol which in replication for survival. beneficial be also may 8-oxo-dGTP rate to incorpo of IV pol ability the dation, to oxi ~100% promotes resistance and by IV pol upregulates response RpoS the exposure to antibiotics. to exposure incorporation. 8-oxo-dGMP and TLS, RDR, including cells, stressed in tolerance DNA damage in multiple functions to perform selected tuting D-loop extension in vitro. in extension D-loop tuting by toreconsti promoteof RDR IV pol ability the examined we directly studies, recent In lacking. been has activity for this evidence biochemical direct cells, stressed in RDR in IV pol implicated strongly 18 E. coli E. Although previous genetics data have have data genetics previous Although Pol IV Preferentially Extends Extends Preferentially Pol IV clamp, which confers processivity confers which β clamp, Taken together, pol IV was probably was Taken together, IV pol D-Loops during Stress D-Loops cells encode for 5 DNA polymer encode cells 19,20 Although the mode of mode the Although 18 Considering that that Considering 18 This activity of activity This 21 . These recent recent . These Hence, the the Hence, E xtr 21 These These a V 2559 i ew . ------

©2013 Landes Bioscience. Do not distribute. 2560 outcompetes pol II at D-loops at such high high atsuch atD-loops II pol outcompetes further IV pol that we found surprisingly, IV. pol and II pol Not between petition com affected concentration in increase relative this whether we examined cells, stationary-phase in ~5000 molecules/cell by ~2-fold IV pol to to upregulate known is response stress RpoS the Since RDR. during errors IV-induced pol proofread and with to compete pol II by enabling mutagenesis adaptive suppresses II pol of domain the that suggest findings our mutations, (~85%) of these promotes most IV pol and vivo in genesis muta adaptive reduces of II pol domain for replicating the . the for replicating responsible are and activities proofreading and synthesis DNA high-fidelity exhibit pol III . III pol multi-subunit the including at D-loops, DNA polymerases other outcompetes stress, during polymerase abundant most We IV, pol the is that found cells. which stressed in polymerases of these trations concen relative using RDR reconstituting by vitro in conditions SOS-induced eled we mod competition, into such insight damage response. damage DNA SOS the during fore upregulated there are and tolerance DNA damage in RDR. error-prone with during compete IV pol to II pol enables function exonuclease this and atD-loops, stimulated is II pol of activity exonuclease the that we found activities. exonuclease and synthesis DNA fidelity high- exhibits that member a B-family is II pol whereas lesion DNA polymerases, of Y-family trans the among Vare and IV (~2500 molecules/cell). ata~7-foldpresent concentration higher IV, pol is with compete which partially able to is at~350present molecules/cell, in stressed cells. stressed in at D-loops IV pol with compete III and II fork. lication rep of the wake the in strand lagging the on maturation fragment Okazaki motes pro enzyme, aDNA repair considered is Pol I, which synthesis. lagging-strand and leading- nt/s) (~600 high-speed performs it where replisome, the within functions past certain lesions. certain past replication performs but also restart, tion Genetic data have implied that pol I, pol that implied have data Genetic

21 Considering that the exonuclease exonuclease the that Considering 22 Pol II is implicated in replica in Pol implicated is II 25 13 21 To gain mechanistic To mechanistic gain As stated above, pol above, stated As Pol II, which is only only is Pol which II, 13,23 Pol I and pol III Pol III pol Iand 21 Unexpectedly, 24 Pol III ------D-loops may be a direct result of RecA. of RecA. result adirect be may D-loops D-loops. RecA-mediated error-prone highly at is IV pol that strate demon findings previous these summary, In respectively). 4, 2and (lane dCTP and of dTTP presence the in as such tides multiple nucleo to incorporate ability its by indicated as template, aprimer with on compared aD-loop extension match more is prone to mis IV pol show that ( poration misincor dNTP in 5-fold increase mate approxi an exhibits IV pol that we found template, primer the as context sequence identical with substrate aD-loop using phates (dNTPs) ( (dNTPs) phates incorrect deoxy-ribonucleoside triphos incorporating against of discrimination degree high arelatively exhibits IV pol that template we found on a primer example, pol II proofreading activity during RDR. during activity proofreading II pol to suppress appear which concentrations fidelity of DNA polymerases ( polymerases of DNA fidelity the to measure used traditionally is which template, aprimer with compared D-loop moreerror-prone ata significantly is IV likely facilitates stress-induced mutations. stress-induced facilitates likely which cells, growth-limited in D-loops roleextending in adominant plays IV pol that indicate findings recent our Overall studies. in recent D-loops IV at polof fidelity the examined we directly RDR, during tions muta promotes stress-induced IV pol that Pol IV is Error-Prone at D-Loops at Error-Prone is Pol IV Nat Struct Mol Biol as a D-loop ( a D-loop Figure 2. The mutagenic activity of pol IV at of IV pol activity mutagenic The Since previous genetic data indicate indicate data genetic previous Since T , G , C, or A, at the top of the gel. gel. the of top the at A, or , C, 21 Strikingly, we found that pol pol that we found Strikingly, Pol IV is error-prone at D-loops. Pol IV activity was analyzed on a primer template ( template a primer on analyzed was activity IV Pol D-loops. at is error-prone IV Pol B Fig. ) under similar conditions in the presence of the indicated dN indicated the of presence the in conditions similar ) under

2 C , right). The results also also results The , right). 2013; 20:748–55. ell Cycle Fig.

2 , l

eft). In contrast, contrast, In eft). Fig. RE , r

2 elative extension. Figure adapted from Pomerantz Pomerantz from adapted Figure extension. elative ) . For 21 ------

rect dNTPs. rect incor against to disciminate ability its reduce potentially and polymerase the of structure the alter conceivably could IV, pol with which interacts RecA that shown have studies previous For example, process referred to as the D-loop cycle. D-loop the to as referred process D-loop, of a the promote dissociation to recombinase of the ability due to the unstable are D-loops RecA-mediated sis. synthe contribute toDNA low-fidelity may D-loops mediated of RecA instability RDR. Consistent with this idea, several several idea, this with Consistent RDR. error-prone highly in resulting extension, D-loop during template strands switch to for DNA polymerase allow would D-loop. of This the strand displaced the within sequence complementary available to anneal transiently may strand invading 3 of the portion unwound the For example, template switching. in result conceivably could strand invading the 3 Transient of the melting 3 of the dissociation partial in result would which strands, parental of the rewinding promote and D-loop the against force ing oppos exert may stress superhelical This D-loop. the from downstream supercoils positive in donor results duplex, which plasmid of the unwinding partial requires formation D-loop For instance, DNA. donor of the topology by the compromised duplex. formed newly the invading by D-loop of the promotes dissociation turn, in that, ssDNA displaced the along a filament forms RecA invasion, strand via formed is D-loop the after For example, end of the invading strand (primer). (primer). strand invading ′ end of the The stability of D-loops may also be be also may of D-loops stability The 26 Alternatively, the inherent inherent the Alternatively, T P s ubstrate, abbreviated abbreviated ubstrate, V olume 12 Issue16 terminus of ′ terminus end of the ′ end of the RT A , e ) and ) and t al. t al. 27 - - -

©2013 Landes Bioscience. Do not distribute. www.landesbioscience.com events. template-switching involves error-prone highly and is (BIR) replication break-induced called of form RDR tract a long that demonstrate of yeast studies prone on the D-loop compared with the the with prone compared D-loop on the error- highly is pol I that ingly, we find Strik template. primer the as context sequence same the containing a D-loop pol I of on fidelity the analyzed we next conditions, identical nearly Using left). (T; Fig. dNTP correct the with pared com as dNTPs incorrect incorporating against to discriminate by ability its cated indi as template, primer on the accurate relatively Iis pol studies, to previous lar Simi analyzed. was sion primer of the exten then s, for 30 added Iwas pol Next, of βonto DNA. assembly in resulting ATP, and dNTP indicated the with along DNA to the protein, were added SSB, binding ssDNA the and clamp-loader, γ the βclamp, The template. pol I of on a primer fidelity the examined acontrol, we as First, used. was fragment, Klenow as known activity, exonuclease 5 Ilacking pol assays following the and 3 and and 5 synthesis DNA high-fidelity exhibits that DNA polymerase A-family ( IV pol with study previous our in as assay same the using D-loop pol of Iata fidelity the we investigated D-loops, mediated RecA to error-prone intrinsic whether is RDR To reaction. the determine catalyzing DNA polymerase of the prone regardless error- is D-loops atRecA-mediated thesis D-loop ( D-loop Figure 3. Pol I is Error-Prone at D-Loops at Error-Prone Pol Iis The possibility exists that DNA syn that exists possibility The ′ –5 Fig. B Pol I is error-prone at D-loops. Pol I activity was analyzed on a primer template ( template aprimer on analyzed was Iactivity Pol D-loops. at Iiserror-prone Pol ′ exonuclease functions. ) under similar conditions in the presence of the indicated dN indicated the of presence the in conditions similar ) under

3 ). Unlike pol IV, pol Unlike an ). Iis pol

-complex 24,31 11,28-30 ′ ′ In In –3 –3

3 ′ ′ ------, pol I. pol by extension mismatch inefficient onstrate dem that studies to previous contrast in is observation This extension. mismatch to promote ability the demonstrates which cases, all in by multiple steps strand ing lane with 3–5 lanes (compare dNTPs incorrect of the presence in more efficient 20–60% by Iis pol extension D-loop that reveals DNA products of the analysis Close ( present is dNTP of which regardless manner, a similar with strand invading the to extend enzyme of the ity abil by the indicated as dNTPs, incorrect incorporating against to discriminate D-loop, however, On the Ifails pol left). (T; Fig. dNTP correct the with pared com dNTPs of incorrect presence the in extension of primer low efficiency tively arela Iexhibits pol template, primer the cally error-prone. cally intrinsi is intermediates recombination at RecA-mediated activity polymerase DNA that indicate findings our D-loops, error-prone highly at also is IV pol that synthesis. DNA high-fidelity to exhibit shown been consistently Ihas pol that considering when especially surprising, was at D-loops error-prone highly Iis pol that observation the Overall, clamp-loader. and β clamp of the presence the in even D-loops ated medi RecA to recruited not efficiently are DNA polymerases or that accessible, not is very strand) (invading primer the 2 Fig. (see study recent our in IV pol with were obtained results Similar added. is (T) dNTP correct the wheneven efficient notby pol I is very primer template ( primer

2). Moreover, pol I extends the invad Moreover, the 2). Iextends pol 31-34 We note that D-loop extension We extension D-loop note that 33-35 Since our recent study shows shows study recent our Since C ). This suggests that either either that suggests This ). ell Cycle T Fig. P . RE

3 , r ) elative extension. elative

. For example, on . For example, Fig.

3 A , right). , right). ) and a ) and

3 ------, error-prone vitro. in atD-loops highly is IV pol that demonstrate ings find recent our and cells, growth-limited in RDR during mutations stress-induced error-prone promotes most polIV Y-family coli E. In of life. domains all in repair HR for mutagenic implications have may error-prone, are findings our eukaryotes and bacteria in of RDR forms certain that demonstrate of evidence lines several Since D-loops. error-prone atRecA-mediated intrinsically is activity DNA polymerase that indicate findings recent our with along replication (BIR) in budding yeast. budding in (BIR) replication break-induced and conversion gene during pol replicative B-family high-fidelity idea, this with Consistent DNA polymerases. among universal is activity RDR mutagenic that IV, Iand pol suggests which like D-loops error-prone also is at pol II that indicates RDR mechanisms in bacteria and eukary and bacteria in mechanisms RDR contributeto high-fidelity normally ways or path factors what is consideration tant impor of an form DNA repair, accurate RDR. mote accurate pro normally that of factors absence the in as such conditions prone particular under error- intrinsically be may RDR that gests sug This templates. on primer synthesis DNA to promote accurate ability their of regardless homologous recombination promote during mutations polymerases DNA high-fidelity cases both in and otes eukary and bacteria in documented well been error-proneAltogether, has RDR of(ALT). telomeres lengthening native alter called process cells—a mammalian and yeast in attelomeres occurs BIR also genic RDR in growth-limited cells. growth-limited in RDR genic to promote shown muta been has activity proofreadingpolymerase possesses that DNA B-family ahigh-fidelity is which II pol Paradoxically, stress. during enzyme of this of upregulation due tobe alack may which mutagenesis, stress-induced promoting for Iin pol no evidence is there study, present the in currently at D-loops error-prone highly also Iis pol we show that cells under stress, which suppresses the the suppresses which stress, under cells stationary-phase in downregulated is coli of E. case the In otes. Perspectives on Error-Prone RDR on Error-Prone Perspectives The data presented in this Extra View View Extra this in presented data The Since HR primarily functions as an an as functions primarily HR Since δ promotes low-fidelity DNA synthesis synthesis promotesDNA low-fidelity , mismatch repair repair , mismatch 21 Although Although 11,28,29,37 36 This This 2561 ------,

©2013 Landes Bioscience. Do not distribute. 2562 BIR. during atD-loops loaded to be likely is helicase MCM hexameric replicative the E. coli E. proliferating in restart of replication case the in example, For RDR. of fidelity rolethe in important playan also may DNA D-loops. error-pronesuppress by stabilizing RDR may proteins mediator recombination example, For RDR. ute to high-fidelity contrib also may D-loops act that factors cells. proliferating in prone RDR error- suppress conceivably could which formation, D-loop during recombination to prevent illegitimate necessary be may pathway this Thus, DNA recombination. of to promote fidelity the functions also differ during error-prone pathways. during RDR differ and mechanisms howsuch RDR fidelity high- for ensuring necessary are pathways and factors what to reveal likely are studies in in RDR in implicated helicase—is SF1 type view, UvrD—a this with Consistent case. heli DnaB replicative the notmay utilize of mode replication particular this Thus, stress. during non-processive to be likely is reduce the fidelity of RDR. of fidelity the reduce conceivably could pathway, which this in function also may helicases processive non- potentially other that ity, suggesting of activ MCM absence the fold in ~4–5 is likely to be non-processive. to be likely is eukaryotes in of mode replication general Drosophila and yeast in pathways RDR during occur switching DNA polymerase and switching plate erated during RDR. during erated gen errors of DNA synthesis correction E. coli, E. growth-limited in D-loops at DNA polymerases between exists tion competi that indicate studies genetic since For example, different. very to be appears coli E. stressed in of RDR mechanism The D-loops. stabilizing tially poten and DNA synthesis processive ing promot by conditions non-stressed under RDR of fidelity contributeto the may tive DNA polymerases, these respec their with conjunction in processive highly are helicases replicative that ering of the newly formed replisome. formed newly of the DNA ahead downstream the unwinds and atD-loops loaded ssDNA—is encircles that ring hexameric helicase—a DnaB tive Replication and recombination co- recombination and Replication Deinococcus radiodurans Deinococcus

40 However, BIR is only decreased by However, decreased only is BIR 1,2,9 cells, the replica the cells, , respectively, this this respectively, , Mismatch repair repair Mismatch cells, however, however, cells, 40 . 39 Since tem Since In yeast, yeast, In 28,41 38 Consid Future 25 RDR RDR ------labeled with with labeled 5 was to room temp. primer ing The cool slowly then °C to 90–100 by heating of RP312 RP313 and followed centrations con equimolar by mixing assembled was template primer The by phosphorimager. visualized and gel polyacrylamide urea adenaturing in were resolved Products 25EDTA. and mM of 45% formamide addition by the were terminated Reactions s. 30 additional for an fragment) (Klenow of pol I 100 addition nM by the followed DTT, 15 at37 for 1min °C MgCl) mM pH 7.5, TRIS-HCl 1mM BSA, 0.1 mg/ml A(25 mM buffer in dNTP indicated (RP312/313) 50plate μ and and 1.3and μ β nM 500 incubating tended and extended DNA products. extended and tended unex of the intensities of the sum by the DNA product extended of the intensity the by dividing determined was extension of DNA Fraction 2. lane in observed that by lane each in observed DNA extension of fraction the by dividing determined was ously described. previ as manner asimilar in performed was IV pol with template extension Primer template. primer of the prior to assembly Biolabs) England (New nucleotide products were purified twice through through twice purified were products 15–30Reaction min. for afurther bated incu SDS and 0.6% and K, proteinase ml 2mg/ EDTA, ofmM 20 addition by the were terminated Reactions s. for 30 added then of concentration was Pol I final nM ATP, μ 0.2 10with μ mixed was reaction 1.5 the Next, min. 1 ATP, and mM phosphocreatine, mM 40 pRP27, supercoiled 0.5 nucleotides) (in 5μ with mixed then was reaction The A for 5min. volume of 5μ atotal in dNTP μ 200 and phokinase, 1μ phosphocreatine, mM 5.2 μ with incubated (RP192) was ssDNA labeled and 1μ and μ Primer extension was performed by performed was extension Primer extension template Primer Sixteen Sixteen extension D-loop g creatine phosphokinase for a further for afurther phosphokinase g creatine l of buffer A containing 740 μ Acontaining l of buffer M M SSB with 40 nM primer tem primer nM 40 with M SSB l of buffer A containing 0.5 A containing l of buffer M RecA, 0.5 ATP, mM M RecA, 40 μ M β M (in nucleotides) nucleotides) M (in 2 32 for 1 min. One hundred One hundred for 1min. γ Methods P- C 21 -complex, 2.6 μ 2.6 -complex, ell Cycle Relative extension (RE) (RE) extension Relative γ -ATP using T4-ATP using poly , 100 nM γ , 100 nM M of the indicated indicated M of the

g creatine phos g creatine M of the M of the l of buffer l of buffer -complex, M SSB, M SSB, 32 ′

-end -end mM mM P-5 M ′ ------used were as previously described. previously as were used (RP192, RP312, RP313) oligonucleotides above. described as determined chased as previously described. previously as chased disclosed. were interest of conflicts No potential 4. 2. muta adaptive responsively: Evolving SM. osenberg 1. 3. described. previously as manner asimilar in formed per was IV pol with extension D-loop by phosphorimager. were analyzed ucts gels. Reaction polyacrylamide urea prod denaturing in resolved then Healthcare) (GE columns HR S-400 microspin Medical Institute. Medical Hughes ES012259), Howard the and (GM21422 MFG and (GM38839), MEO (R00CA160648), to RTP (grants of Health Institutes National 7. 6. 5. Disclosure of Potential Conflicts of Interest of Conflicts ofPotential Disclosure

Plasmid (pRP27) and synthetic DNA synthetic and (pRP27) Plasmid DNA All proteins were prepared or pur prepared were proteins All Proteins This work was supported by the US by the supported work was This 2009; 191:5881-9; JBacteriol chromosome. coli Escherichia the in tions muta stationary-phase of sequences and SM. Stress-induced beta-lactam antibiotic resistance org/10.1080/10409230701648502 42:399-435; 2007; Biol Mol Biochem Rev Crit evolvability. of regulation the and response astress as Mutation 42:373-97; 2007; Biol Mol Biochem Rev Crit teria. http://dx.doi.org/10.1038/35080556 PMID:11433357 2:504-15; 2001; Genet Rev Nat tion. P G org/10.1080/10409230701648494 F R org/10.1073/pnas.1104681108 108:13659-64; USA2011; Sci Acad Natl Proc coli. Escherichia in mutation on breaks DNA of repair mutagenic to switch astress-inducible of Impact SM. Rosenberg dx.doi.org/10.1371/journal.pbio.0030176 PMID:15869329 3:e176; 2005; Biol PLoS resistance. antibiotic of evolution the combating and mutation of Inhibition FE. WA, Romesberg Craig org/10.1128/JB.00732-09 http://dx.doi.org/10.1126/science.1082240 PMID:12775833 300:1404-9; 2003; Science teria. bac in mutagenesis Stress-induced al. et M, Radman B S C hee C, Gibson JL, Darrow MC, Gonzalez C, C, Gonzalez MC, Darrow JL, Gibson C, hee etrosino JF, Galhardo RS, Morales LD, Rosenberg Rosenberg LD, Morales RS, JF, Galhardo etrosino oster PL. Stress-induced mutagenesis in bac in mutagenesis Stress-induced PL. oster jedov I, Tenaillon O, Gérard B, Souza V, Denamur E, E, V, Denamur Souza B, O, Gérard Tenaillon I, jedov irz RT, Chin JK, Andes DR, de Crécy-Lagard V, Crécy-Lagard de DR, Andes JK, RT, Chin irz alhardo RS, Hastings PJ, Rosenberg SM. SM. PJ, Rosenberg Hastings RS, alhardo 21 Relative extension (RE) was was (RE) extension Relative PMID:17917873 Acknowledgments PMID:17917874 PMID:21808005 References PMID:19648247 V olume 12 Issue16 http://dx.doi. ; http://dx.doi. ; http://dx.doi. ; http://dx.doi. ; 21 21 http:// ; ------; ;

©2013 Landes Bioscience. Do not distribute. 13. 19. 18. 17. 16. 15. 14. www.landesbioscience.com 12. 11. 10. 9. Hastings MJ, Lombardo PL, GJ, Lee cKenzie 8.

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