A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion- stalled replisomes

Seungwoo Changa,1, Karel Naimanb,1,2, Elizabeth S. Thralla, James E. Katha, Slobodan Jergicc,d, Nicholas E. Dixonc,d, Robert P. Fuchsb,3,4, and Joseph J. Loparoa,4

aDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; bTeam DNA Damage Tolerance, Cancer Research Center of Marseille, CNRS, UMR7258, F-13009 Marseille, France; cSchool of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW 2522, Australia; and dMolecular Horizons, University of Wollongong, Wollongong, NSW 2522, Australia

Edited by Michael E. O’Donnell, HHMI and The Rockefeller University, New York, NY, and approved November 5, 2019 (received for review August 21, 2019) DNA lesions stall the replisome and proper resolution of these genetically tractable while retaining the same basic architecture of obstructions is critical for genome stability. Replisomes can directly more complicated systems (2, 3). In E. coli the majority of lesion- replicate past a lesion by error-prone translesion synthesis. Alterna- stalled replisomes are resolved through DA pathways, particularly tively, replisomes can reprime DNA synthesis downstream of the when the SOS damage response is not induced. However, upon lesion, creating a single-stranded DNA gap that is repaired primarily induction of the SOS response and the concomitant increase in in an error-free, homology-directed manner. Here we demonstrate TLS polymerase levels, higher fractions of stalled replisomes are Escherichia coli how structural changes within the replisome deter- resolved through TLS (4, 5). mine the resolution pathway of lesion-stalled replisomes. This path- E. coli cells have 3 TLS polymerases, Pol II, IV, and V (5, 6). way selection is controlled by a dynamic interaction between the Among these, Pol II and IV are abundant even before their proofreading subunit of the replicative polymerase and the proces- expression levels are highly elevated during the damage-induced sivity clamp, which sets a kinetic barrier to restrict access of trans- SOS response. If TLS polymerases gained frequent access to the lesion synthesis (TLS) polymerases to the primer/template junction. extending primer, replication would be severely inhibited due to Failure of TLS polymerases to overcome this barrier leads to reprim- the much slower polymerization of TLS polymerases as com- BIOCHEMISTRY ing, which competes kinetically with TLS. Our results demonstrate – that independent of its exonuclease activity, the proofreading sub- pared to Pol III, the replicative polymerase (7 9). Intriguingly, unit of the replisome acts as a gatekeeper and influences replication despite the high abundance of Pol II and Pol IV relative to the fidelity during the resolution of lesion-stalled replisomes. replicative polymerase both in the SOS response-uninduced and -induced cells, TLS polymerases only modestly inhibit replication DNA replication | replication stalling | translesion synthesis | damage avoidance | repriming Significance

enomic DNA is constantly damaged by various intracellular DNA replication is the high-fidelity process by which cells dupli- Gand extracellular agents. Replication is transiently blocked cate their chromosomes prior to cell division. Cellular DNA is at these sites because replicative DNA polymerases are generally constantly damaged, and the resulting DNA lesions can block poor at synthesizing past lesions. Stalled replisomes are resolved replication, leading to genome instability and cell death. Cells use primarily through either recombination-dependent damage avoid- multiple pathways to resolve stalled replication. Understanding ance (DA) pathways or translesion synthesis (TLS), and dis- resolution pathway choice is important because some of these tinct DNA intermediates are created during each process (1). pathways are more likely to introduce mutations than others. In In DA pathways, damaged templates are replicated in an error- bacterial cells, replication stresses, including antibiotic treatment, free manner using an undamaged homologous sister chromatid as lead to mutagenesis, which may contribute to the emergence a template via processes that utilize homologous recombination of antibiotic resistance. In this study, we describe a molecular factors. In contrast, during TLS damaged templates are directly interaction within the Escherichia coli replication machinery that replicated by TLS polymerases in an error-prone manner. TLS can plays a crucial role in resolution pathway choice, thereby influ- occur through sequential polymerase switching between the rep- encing whether lesion-stalled replication is resolved in an error- licative polymerase and a TLS polymerase, yielding a continuous prone or error-free manner. DNA product (TLS at the fork). Alternatively, replisomes can reprime DNA synthesis downstream of the lesion leaving a single- Author contributions: S.C., K.N., E.S.T., R.P.F., and J.J.L. designed research; S.C., K.N., E.S.T., R.P.F., and J.J.L. performed research; S.C., K.N., E.S.T., J.E.K., S.J., N.E.D., R.P.F., and J.J.L. stranded DNA (ssDNA) gap behind. These gaps are then filled in contributed new reagents/analytic tools; S.C., K.N., E.S.T., R.P.F., and J.J.L. analyzed data; either by TLS polymerases (gap-filling synthesis) or by homology- and S.C., K.N., E.S.T., J.E.K., S.J., N.E.D., R.P.F., and J.J.L. wrote the paper. dependent gap repair (HDGR) (1). Given the marked differences The authors declare no competing interest. in mutagenic potential between DA pathways and TLS, it is im- This article is a PNAS Direct Submission. portant to understand what determines pathway choice at stalled Published under the PNAS license. replisomes. 1S.C. and K.N. contributed equally to this work. Structural changes within the replisome upon lesion stalling, 2Present address: Genome Damage and Stability Centre, School of Life Sciences, University such as conformational changes of replisome components and of Sussex, BN1 9RQ Falmer, United Kingdom. – alterations in protein protein interactions, likely play an important 3Present address: Marseille Medical Genetics, UMR1251 Aix Marseille Université/Inserm, role in pathway selection, yet these dynamics have been largely 13385 Marseille, France. unexplored. DNA replication of Escherichia coli serves as an at- 4To whom correspondence may be addressed. Email: [email protected] or tractive model system to probe these lesion-induced structural [email protected]. changes of the replisome as it uses both DA pathways and TLS This article contains supporting information online at https://www.pnas.org/lookup/suppl/ to resolve stalled replisomes. Moreover, the E. coli replisome can doi:10.1073/pnas.1914485116/-/DCSupplemental. be reconstituted with a relatively small number of factors and is

www.pnas.org/cgi/doi/10.1073/pnas.1914485116 PNAS Latest Articles | 1of11 Downloaded by guest on September 26, 2021 in E. coli cells (10) and they contribute little to spontaneous (2, 32, 33). Replication of the lesion-free control template by the mutagenesis (11, 12). Collectively, these observations suggest reconstituted E. coli replisome resulted in the rapid formation of that TLS polymerases are largely excluded from replisomes replication products that can be resolved by denaturing gel elec- (13, 14). trophoresis: A resolution-limited leading-strand product band The E. coli β2 clamp processivity factor plays an important role and a distribution of smaller lagging-strand products along in regulating TLS because all TLS polymerases must bind the β2 with a fraction of unreplicated templates (Fig. 1B and SI Ap- clamp to perform TLS (5, 9, 15–17). The β2 clamp is a homo- pendix,Fig.S1B) (2). The resolution limit (RL) of our gel is ∼45 dimer that encircles DNA and tethers DNA polymerases to their kilonucleotides, and therefore accumulation of leading-strand template (18, 19). Each protomer of the β2 clamp molecule has a products at the RL indicates that at least 6 cycles of rolling- hydrophobic cleft, a common binding site for clamp binding circle replication occurred on each template. Consistent with a 2 proteins, yielding 2 identical clefts per β2 clamp molecule (20). prior observation that a single N -FFdG blocks primer exten- Clamp binding proteins have 1 or more clamp binding motifs sion by Pol III (9), replication of the N2-FFdG–containing (CBMs) that interact with the β2 clamp via cleft–CBM interactions template in the absence of Pol IV was strongly attenuated by a (21, 22). All 5 E. coli DNA polymerases have 1 or 2 conserved single N2-FFdG on the leading-strand template (Fig. 1B). We CBMs (17, 21, 23). Pol III is a trimeric complex (αeθ) consisting of also observed faint, discrete bands between resolution-limited polymerase (α), exonuclease (e) and accessory (θ) subunits. The α replication products and unreplicated templates (Fig. 1 B and C). subunit has an internal CBM, which is required for processive As these products were created only in the presence of N2-FFdG, replication (24). In addition, the e subunit has an internal CBM, they represent lesion-stalled replisomes that have undergone dif- which is responsible for the replication-promoting role of the e ferent multiples of replication around the template with TLS over subunit (25–27). During processive replication, the α and e sub- the lesion inefficiently mediated by Pol III (34). units of the Pol III complex occupy both clefts of a β2 clamp To determine whether Pol IV-mediated TLS might resolve molecule (26–28). However, unlike the internal CBM of the α this stalling, we examined the effect of Pol IV on replication of subunit, the CBM of the e subunit has a relative low binding af- the N2-FFdG–containing template. Upon addition of increasing finity to the cleft; this results in its frequent detachment from a amounts of Pol IV, synthesis of both the leading and lagging cleft, causing temporary pauses during processive replication (29, strands was gradually restored and long leading-strand replica- 30). Given the dynamic nature of the e subunit–β2 clamp in- tion products accumulated at the RL before replication was fully teraction, it may constitute a key factor in regulating the access of inhibited by Pol IV at high concentrations (Fig. 1C and SI Ap- cleft-binding proteins, such as TLS polymerases. pendix, Fig. S1E) (7). The robust formation of these resolution- Here we show that dynamic interactions between Pol III and limited products shows that TLS occurs efficiently over the lesion the β2 clamp dictate the fate of lesion-stalled replisomes. We at the fork as repriming of leading-strand synthesis would result demonstrate that when Pol IV is present at optimal levels, TLS in a gap that terminates rolling circle replication. This termina- occurs at the fork through sequential polymerase switching be- tion results from the displacement of the circular leading-strand tween Pol III and Pol IV. In contrast, when Pol IV is present at template that occurs when the helicase runs into the strand suboptimal levels, a higher fraction of lesion-stalled replisomes is discontinuity resulting from repriming (SI Appendix, Fig. S1F). resolved by repriming replication downstream, leaving an ssDNA Consistent with the requirements of Pol IV-mediated TLS in gap. We show that besides its canonical proofreading function, cells, the ability of Pol IV to promote replication of the N2-FFdG– the e subunit plays a gatekeeping role that largely prevents Pol containing template required both its catalytic and clamp-binding IV from replacing Pol III during processive replication and limits activities (SI Appendix,Fig.S1D, E,andG) (17). Pol IV-mediated the usage of TLS to resolve lesion-stalled replisomes. Central to TLS occurred less efficiently over an N2-(1-carboxyethyl)-2′- this gatekeeping role is the dynamic interaction between the e deoxyguanosine (N2-CEdG) lesion and was strongly blocked by subunit and the β2 clamp. When this interaction is strengthened tetrahydrofuran (THF) (SI Appendix,Fig.S1H), indicating that in vitro, Pol IV-mediated TLS at the fork is suppressed, leading our rolling-circle assay is sensitive to the efficiency by which Pol to more lesion skipping. Conversely, when the e–β2 interaction is IV mediates TLS over various lesions (35, 36). weakened, Pol IV more efficiently mediates TLS at the fork and Moreover, at optimal Pol IV concentrations, replication of processive replication is more readily inhibited by Pol IV. In the N2-FFdG–containing template, as measured by quantifying cells, when the SOS-response is not induced, TLS by all 3 TLS leading-strand replication products (SI Appendix,Fig.S1C), was polymerases is minimally employed to resolve lesion-stalled slowed only by ∼40% compared to replication of the control replisomes. However, weakening the e–β2 clamp interaction template (SI Appendix,Fig.S1I). Given that the polymerization leads to resolution of a much larger fraction of lesion-stalled rate of the Pol IV-based E. coli replisome is ∼10 nt/s (7, 9), which replisomes through TLS, indicating that the strength of the is over 50 times lower than that of the Pol III-based replisome, e–β2 interaction is tuned to suppress TLS in uninduced cells. these results indicate that Pol IV only briefly switches with Pol III Collectively, these results show that the e–β2 interaction sets a (9, 37). Collectively, these results demonstrate that within our kinetic barrier to the access of TLS polymerases to the extending reconstitution, Pol IV can efficiently mediate TLS at the replica- primer and thus suppresses TLS. tion fork by switching with Pol III, synthesizing a small patch of DNA over the lesion. Results Rapid and Efficient Pol IV-Mediated TLS within Lesion-Stalled The e–Cleft Contact Mediates Polymerase Exchange during TLS. As Replisomes. In an effort to determine how processive replication Pol III core (αeθ) occupies both clefts of the β2 clamp during and TLS are coordinated, we reconstituted Pol IV-mediated TLS processive replication (26) and Pol IV must bind to a cleft for its on a rolling-circle DNA template that contains a site-specific N2- action, we sought to determine how Pol III disengages from the furfuryl dG (N2-FFdG) lesion on the leading-strand template (Fig. clamp to allow for Pol IV to mediate TLS at the fork. To address 1A and SI Appendix, Fig. S1A). N2-FFdG is an attractive model this question, we varied the strength of the interaction between 2 lesion because Pol IV is proficient at replicating past N -dG ad- the Pol III core and the β2 clamp and examined the effect on ducts and structurally related DNA lesions can be created in living TLS. The α and e subunits of the Pol III core (αeθ) each occupy a cells by treatment with nitrofurazone (NFZ) (31). In rolling-circle cleft via independent CBMs, albeit with vastly different affinities; replication, replication proceeds over the circular template mul- the e–cleft contact is ∼250-fold weaker than the α–cleft in- tiple times, generating a long leading-strand tail that serves as teraction (Fig. 2A) (24, 26). Replacing the wild-type CBM of the a template for discontinuous lagging-strand synthesis (Fig. 1A) e subunit with a mutant CBM (eL), which binds the cleft ∼500

2of11 | www.pnas.org/cgi/doi/10.1073/pnas.1914485116 Chang et al. Downloaded by guest on September 26, 2021 Assembly Initiation Quenching times tighter than the wild-type CBM (26), suppressed Pol IV- A mediated TLS compared with the wild-type Pol III core (αeθ) ‘ SSB  DnaG and required higher Pol IV concentrations for optimal TLS (Fig.

2 rNTPs 2B). Normalizing replication of the lesion containing template by DnaB dATP/dTTP 6 replication of the lesion-free control template (SI Appendix, dCTP/dGTP [-32P]-dATP Pol IV Supplementary Methods) showed that the «L mutation suppressed Pol IV-mediated TLS to ∼40% (Fig. 2 B, Right Inset). Strengthening ~7.2 kbp the interaction also modestly enhanced replicative activity on a 5’ lesion-free template (Fig. 2 B, Left Inset) (26). These results suggest Leading strand template Leading that the disengagement of the e subunit from the β cleftatalesion Lagging strand template Lagging 2 is necessary for Pol IV to bind to the β clamp and perform TLS. N2-furfuryl dG (N2-FFdG) O 2 As the α subunit also contains a CBM, we next considered if N NH disengagement of α was additionally required for Pol IV-mediated O N TLS (Fig. 2A). To address this possibility, we examined the effect N N H HO of strengthening the α–cleft interaction within our biochemical O reconstitution. Unlike the effects of strengthening the e–cleft in- H H α H H teraction, replacing the wild-type CBM of the subunit with a OH H mutant CBM (αM3), which binds the cleft ∼100 times tighter than the wild-type CBM (24)—even ∼50 times tighter than the eL–cleft B Control dG N2-FFdG interaction—had little effect on replication of the lesion-free or 0.5 1.5 3 5 9 15 0.5 1.5 3 5 9 15 lesion-containing template (Fig. 2C). Collectively, these results 0 1 2 4 7 12 20 0 1 2 4 7 12 20 demonstrate that disengagement of only the e subunit, not the

Time (min) Time α subunit, within lesion-stalled replisomes promotes Pol IV to bind to a cleft and perform TLS. RL To validate these in vitro observations in cells, we introduced Stalled « αM3 replication the dnaQ( L) and dnaE( ) mutations, individually or in com- bination, into their respective genomic loci and examined their Leading effects on cellular sensitivity to DNA damaging agents NFZ and BIOCHEMISTRY methyl methanesulfonate (MMS) (Fig. 2D). Cells lacking Pol IV

Template (ΔdinB) were strongly sensitized to NFZ and MMS, indicating that Pol IV-mediated TLS contributes to cellular tolerance to these agents. Consistent with our in vitro observations, the dnaQ(«L) strain was sensitized to both NFZ and MMS, whereas the Lagging dnaE(αM3) strain retained wild-type tolerance. Furthermore, M3 the strain containing both the dnaQ(«L) and dnaE(α ) mutations « αM3 « C [dnaQ( L)dnaE( )] resembled the sensitivity of the dnaQ( L) N2-FFdG strain, indicating that the strengthened e–cleft interaction is re- Pol IV sponsible for the increased sensitivity. This increased sensitivity of - [Pol IV] (nM) 39 78 312 2500 625 - « 156 1250 + the dnaQ( L) strain is due to defective Pol IV-mediated TLS be- cause dnaQ(«L) was epistatic to ΔdinB (SI Appendix,Fig.S2A). « αM3 n>6 The dnaQ( L) and dnaE( ) strains grew normally and retained 5 nearly wild-type DNA content (SI Appendix,Fig.S2B and C). RL 4 Furthermore, the dnaQ(«L) strain retained similar numbers of

Leading + 3 replisome foci to the dnaQ strain both in untreated and NFZ- treated cells (SI Appendix,Fig.S2D and E). The dnaQ(«L) and 2 dnaE(αM3) strains also retained nearly wild-type SOS-responses to Leading both NFZ and MMS (SI Appendix,Fig.S2F). Collectively these results rule out the possibility that the increased sensitivity of the Template Stalled replication dnaQ(«L) strain results from a general defect in DNA replication

Template 1 or the DNA damage response.

e Subunit Acts as a Gatekeeper. Because the dnaQ(«L) mutation stimulates proofreading of Pol III core (αeθ) (27, 38), we next addressed whether the eL-containing replisome suppressed TLS Lagging through futile cycles of TLS and proofreading, impeding the transition from TLS to processive replication (SI Appendix, Fig. S3A) (39, 40). If the proofreading function of the e subunit counteracts Pol IV-mediated TLS, abrogating the exonuclease Fig. 1. In vitro reconstitution of Pol IV-mediated TLS. (A) A rolling-circle activity of the e subunit should promote Pol IV-mediated TLS. replication-based TLS assay. Replication reactions were performed Indeed, when the catalytic residues of the e subunit were mutated through the indicated steps. Newly synthesized DNAs were labeled with (αeD12A,E14Aθ) (41), Pol IV-mediated TLS was promoted (Fig. incorporated [α-32P]-dATP during replication, separated on a denaturing 3A), indicating that the proofreading function of the e subunit gel and visualized by autoradiography. (B) A single N2-FFdG in the leading-strand template potently inhibits the formation of RL leading- strand products by the E. coli replisome. Control, a lesion-free template; N2-FFdG, a N2-FFdG–containing template. (C) Pol IV promotes replication Reactions were quenched 12 min after initiation. (Right) Magnified view of of the N2-FFdG–containing template. (Left) Replication of the N2-FFdG– the same gel; −, no Pol IV; +, 156 nM Pol IV; numbers (n), number of passages containing template in the presence of indicated amounts of Pol IV. through a N2-FFdG lesion.

Chang et al. PNAS Latest Articles | 3of11 Downloaded by guest on September 26, 2021 Control dG N2-FFdG A α C 625 625 39 78 39 78 312 - - 312 156 [Pol IV] (nM) 156

αεθ -cleft Pol IV-cleft interaction RL ε 90° αM3εθ Pol IV β2 1.4 -cleft 1.5 1.5 1.2 Cleft 1.0 1.0 RT wild-type strengthened 1.0 RR 0.5 0.5 Fold Sequence K (μM) Sequence K (μM) 0.8 0.0 0.0 D D strenthening αεθ αM3εθ αεθ αM3εθ 0.6 α subunit 920QADMF924 ~0.8 920QLDLF924 (αM3) ~0.007 ~100 0.4 ε subunit 182QTSMAF187 ~210 182QLSLPL187(ε ) ~0.4 ~500 αεθ αεθ L 0.2 αM3εθ αM3εθ 346 351 Pol IV QLVLGL Rel. Band Intensities 0.0 0 0 100 200 100 200 500 600 700 500 600 700 300 400 300 400 B Control dG N2-FFdG [Pol IV] (nM) [Pol IV] (nM) D NFZ No 8 μM 10 μM 39 312 39 312 78 78 625 [Pol IV] (nM) - 625 - 156 156 Dilution -1 -5 -1 -2 -3 -4 -5 -6 -1 -2 -3 -4 -5 -6 + + + αεθ dnaE dnaQ dinB RL ∆dinB dnaEM3 αεLθ dnaQ(εL) 1.2 dnaEM3 dnaQ(ε ) 2.0 1.5 L 1.0 1.5 1.0 dnaEM3 ∆dinB

1.0 RT RR 0.8 0.5 0.5 MMS No 4 mM 6 mM 0.0 Dilution -1 -5 -1 -2 -3 -4 -5 -6 -1 -2 -3 -4 -5 -6 0.0 αεθ αε θ αεθ αε θ 0.6 L L dnaE+ dnaQ+ dinB+ 0.4 ∆dinB αεθ αεθ M3 0.2 dnaE αεLθ αεLθ dnaQ(ε ) Rel. Band Intensities L 0.0 M3 0 0 dnaE dnaQ(εL) 100 200 500 600 700 100 200 500 600 700 300 400 300 400 dnaEM3 ∆dinB [Pol IV] (nM) [Pol IV] (nM)

Fig. 2. e–Cleft mediates polymerase exchange. (A) Interactions between Pol III core (αeθ) or Pol IV and the β2 clamp via the CBM–cleft interaction. (Upper) α–Cleft, the cleft occupied by the α subunit; e–cleft, the cleft occupied by the e subunit. Pol IV interacts with a cleft via the C-terminal CBM. For simplicity, the θ subunit of Pol III core is not depicted. (Lower) Mutations on CBMs to strengthen the interaction with a cleft. (B) Strengthening the e–cleft interaction sup- presses in vitro TLS. (Upper) RL leading-strand replication products. (Lower) Relative band intensities were calculated with respect to the band intensity in the absence of Pol IV for the lesion-free template and the maximal band intensity for the lesion-containing templates in the presence of Pol IV, respectively; αeθ,

wild-type e-containing replisome; αeLθ, eL-containing replisome. (Inset, Left) Relative replication (RR) corresponds to replication of the control template normalized by replication of the αeθ-containing replisome at [Pol IV] = 156 nM. (Inset, Right) Relative TLS (RT) was calculated by first normalizing replication of the lesion-containing template with replication of the control and then calculating the ratio with respect to the αeθ replisome. Reported values correspond to [Pol IV] = 156 nM (mean ± SD, n = 3). (C) Enhancing the α–cleft contact has little effect on in vitro replication or TLS. (Upper) RL leading-strand replication products. (Lower) Relative band intensities of leading-strand replication products; αeθ, wild-type e-containing replisome; αM3eθ, αM3-containing replisome. (Inset, Left) RR, replication of the control template at [Pol IV] = 156 nM. (Inset, Right) replication-normalized RT at [Pol IV] = 156 nM (mean ± SD, n > 2). (D) Strengthening the e–cleft interaction sensitizes cells to NFZ and MMS. Cultures of indicated strains were serially diluted and spotted on LB-agar plates containing indicated concentrations of either NFZ or MMS. dnaQ and dnaE, the genes encoding the e and the α subunits, respectively.

antagonizes Pol IV-mediated TLS over N2-FFdG, likely through this increase is difficult because weakening the e–cleft interaction futile cycles. In contrast to the effect on TLS, abrogating the impacts not only the access of TLS polymerases but also the exonuclease activity of the e subunit reduced replication on the proofreading activity of Pol III core (27, 38). On the N2-FFdG– control template (Fig. 3A), indicating that exonuclease activity containing template, Pol IV-mediated TLS occurred more effi- facilitates processive replication. Importantly, we found that the ciently in the eQ-containing replisome (αeQθ) compared with the dnaQ(«L) mutation still strongly suppressed Pol IV-mediated wild-type replisome (αeθ) when normalized for replication on the TLS within the exonuclease-dead dnaQ-containing replisome control template (Fig. 3 B, Right Inset). Removal of the e subunit (αeD12A,E14Aθ), which was comparable to the suppression within further increased the efficiency and potency of Pol IV-mediated the wild-type replisome (αeθ)(Fig.3A, Inset and SI Appendix,Fig. TLS (Fig. 3 B, Right Inset), suggesting that in the absence of the e e– S3B). This result indicates that strengthening the cleft interaction subunit, Pol IV may frequently bind to the β2 clampevenduring suppresses Pol IV-mediated TLS by inhibiting the interaction of processive replication. Similar observations were also made 2 Pol IV with the β2 clamp rather than promoting futile cycles of TLS with the N -CEdG–containing template (SI Appendix,Fig. and proofreading. S3C). These results demonstrate that it is a gatekeeping activity Given that these results suggest an exonuclease activity- of the e subunit rather than its exonuclease activity that limits independent gatekeeping role of the e subunit, we hypothesized the access of Pol IV and potentially other clamp-binding pro- that weakening the e–cleft interaction or removing e would make teins to lesion-stalled replisomes. replication more potently inhibited by Pol IV and increase Pol IV- mediated TLS. To weaken the e–cleft interaction, we introduced Strength of the e–Cleft Interaction Determines Pathway Choice the dnaQ(«Q) mutation, which substantially decreases affinity for between TLS at the Fork and Repriming. Given prior observations the clamp (KD > 2 mM) (26). Indeed, the eQ-containing replisome that the reconstituted E. coli replisome can reprime leading-strand (αeQθ) exhibited reduced replicative activity on the control tem- synthesis (42, 43), we next asked if suppression of TLS at the fork plate and replication was more potently inhibited by Pol IV as by strengthening the e–cleft interaction increased repriming. To compared with the wild-type replisome (αeθ)(Fig.3B, Left Inset). determine whether the N2-FFdG–stalled replisome reprimed These results indicate that access of Pol IV to the primer/template downstream of the lesion, we employed Southern blotting to de- (P/T) junction during processive replication is inhibited by the tect the expected repriming products (Fig. 4A and SI Appendix, e subunit. Consistent with this gatekeeping role, spontaneous Fig. S1F). In the absence of Pol IV, replication of the N2-FFdG– mutagenesis was elevated when the e–cleft interaction was weak- containing template created replication products that were de- ened (SI Appendix,TableS1). However, identifying the origin of tected with leading-strand specific Southern blot probes (Fig. 4B

4of11 | www.pnas.org/cgi/doi/10.1073/pnas.1914485116 Chang et al. Downloaded by guest on September 26, 2021 Control dG N2-FFdG and D). These short leading-strand products were better detected A with a distal leading-strand probe (1,901-nt probe) than a proximal 39 625 39 625 78 312 [Pol IV] (nM) - - 78 312 leading-strand probe (40-nt probe) (Fig. 4A and SI Appendix, Fig. 156 156 S4E), consistent with prior reports that repriming occurs a few αεθ hundred nucleotides downstream of the lesion (42). Collectively, these results indicate that replisomes stalled at N2-FFdG reprime αεLθ 2 RL DNA synthesis downstream of N -FFdG, leaving an ssDNA gap between the lesion and a newly synthesized leading-strand RNA αεCDθ primer (Fig. 4A). CD The addition of increasing concentrations of Pol IV to replica- αεL θ tion reactions of the N2-FFdG–containng template led to a grad- 1.4 αεθ 1.5 ual increase in TLS at the fork and a concomitant decrease in 1.2 αε θ repriming, suggesting that Pol IV-mediated TLS competes with L 1.0 1.0 αεCDθ repriming (Fig. 4B and SI Appendix,Fig.S4F–H). Indeed, when αε CDθ 0.5RT 0.8 L wild-type Pol IV (100 nM) was added with varying time delays 0.0 after the initiation of replication, stalled replisomes were rapidly θ θ θ

0.6 L CD CD

αεθ released into the TLS pathway and no further increase in rep- L αε

0.4 αε

αε riming was observed (Fig. 4C). 0.2 Notably, for the eL-containing replisome, while TLS was sup- Rel. Band Intensities 0.0 pressed, repriming persisted in the presence of higher concen- 0 0 trations of Pol IV as compared with the wild-type replisome (Fig. 100 400 700 100 400 700 200 300 500 600 200 300 500 600 e– [Pol IV] (nM) [Pol IV] (nM) 4B). To examine whether strengthening the cleft interaction increases the intrinsic propensity of the replisome to reprime, we Control dG N2-FFdG B compared the time course of repriming in the absence of Pol IV between the wild-type and eL-containing replisomes (SI Appen- 39 625 39 625 [Pol IV] (nM) - 78 312 - 78 312 156 156 dix, Fig. S4I). Both replisomes had a similar partitioning between αεθ Pol III-mediated TLS over the lesion and repriming with rep- riming occurring at similar rates (t1/2,apparent ∼4 min). Therefore,

αε θ RL BIOCHEMISTRY Q the persistent repriming of the eL-containing replisome at higher concentrations of Pol IV most likely results from suppression of α Pol IV-mediated TLS. 1.2 1.2 4 e–β 1.0 αεθ In Vivo, Pol IV-Mediated TLS Increases upon Weakening the 2 1.0 αε θ 3 0.8 Q Clamp Interaction. Our in vitro observations demonstrate that 0.6 α 2 e– NR 0.8 RT the cleft interaction plays a decisive role in determining the 0.4 1 0.6 0.2 fate of lesion-stalled replisomes. To test whether this also hap- 0.0 0 pened in living cells, we employed an in vivo assay that quantita- 0 0.4 αεθ αεQθ α 100 300 700 200 400 500 600 tively measures the fraction of stalled replisomes that are resolved 0.2 [Pol IV] (nM) either by TLS or via the DA pathway at a site-specific lesion lo-

Rel. Band Intensities cated in the chromosome. The experimental system (SI Appendix, 0.0

0 0 Fig. S5 A and B), which is based on phage-λ site-specific recom-

100 200 300 400 500 600 700 100 200 300 400 500 600 700 bination, consists of 2 major components: A recipient E. coli strain [Pol IV] (nM) [Pol IV] (nM) with a single attR site, and a nonreplicating plasmid construct containing the single lesion of interest, an attL site, and an am- Fig. 3. e Subunit suppresses TLS through a gatekeeping role. (A)Strength- picillin resistance gene. A site-specific recombination reaction ening the e–cleft interaction with the dnaQ(«L) mutation suppresses TLS at the fork for both wild-type and catalytically defective e subunits. (Upper) RL leading- between attL and attR is controlled by ectopic expression of phage- strand replication products. (Lower) Relative band intensities of these leading- λ integrase and excisionase proteins (44) and leads to the inte- CD D12A,E14A CD CD strand replication products; e , catalytically defective e (e ); eL , e gration of the lesion-containing plasmid into the chromosome. « with the dnaQ( L) mutation. (Inset, Right)Replication-normalized RT (relative Integrants are selected based on their resistance to ampicillin. The TLS) at [Pol IV] = 78 nM (mean ± SD, n = 2). (B) Weakening the e–cleft inter- chromosomal region where integration takes place carries the action promotes Pol IV-mediated TLS at the fork. RL leading-strand replication 3′-end of the lacZ gene fused to attR (minute 17 in the E. coli products resulting from replication of a lesion-free control template (Upper, ′ 2 – chromosome), while the remaining 5 -end is located on the Left)oraN-FFdG containing template (Upper, Right) by the indicated Pol III incoming plasmid fragment in fusion with attL. Precise integration complexes. (Lower) Relative band intensities of these leading-strand replication + restores a functional β-galactosidase gene (lacZ ). A short se- products. (Inset, Left) Inhibition of replication by Pol IV; replication is normal- “ ” ized to replication in the absence of Pol IV (NR, normalized replication). (Inset, quence heterology inactivates the lacZ gene in the opposite Right) Replication-normalized RT at [Pol IV] = 156 nM (mean ± SD, n > 2); αeθ, strand across from the single lesion and serves as a genetic marker

wild-type replisome; αeQθ, eQ-containing replisome; α, e-free replisome. to allow strand discrimination during replication (45). The recip- ient strain carries both uvrA and mutS deletions to prevent the single lesion and the sequence heterology from being repaired by and SI Appendix,Fig.S4A–C) (2). Replication products that ran as nucleotide excision repair and mismatch repair, respectively. discrete bands above the template were leading-strand replication Functional LacZ is expressed only when the lesion-containing products that resulted from processive replication because these leading-strand template is bypassed by TLS events with the were detected only with leading-strand probes and were longer exception of events in which a frameshift mutation is in- than the template (Fig. 4B and SI Appendix,Fig.S4C). Addition- troduced (SI Appendix,Fig.S5B and C). Therefore, colonies ally, we detected diffuse replication products that ran below the resulting from TLS events appear as blue/white-sectored colo- template. These replication products were not created in the ab- nies on X-Gal–containing plates (46). On the other hand, white sence of DnaG, but unlike Okazaki fragments these were detected colonies result from RecA-mediated HDGR (47). For most blue/ with leading-strand probes (Fig. 4B and SI Appendix,Fig.S4C white-sectored colonies observed, the blue and white sectors were

Chang et al. PNAS Latest Articles | 5of11 Downloaded by guest on September 26, 2021 roughly equal, indicating that in the majority of cases TLS occurred A N2-FFdG Leading strand probes in the first round of replication. RNA primer Lagging strand probes To investigate Pol IV-mediated TLS, we introduced a single (−)-trans-anti-benzopyrene-N2-dG adduct [dG-BaP(−)] into the E. coli genome, known to be bypassed only by Pol IV (48, 49). In + the dnaQ background, the majority of G-BaP(-)-stalled repli- 40 nts somes were resolved by the DA pathway, while only 20% by TLS « 900 nts (Fig. 5A). However, in the dnaQ( Q*) background, which weak- ens the e–cleft interaction, 55% of the stalled replisomes were 1901 nts resolved by TLS with a corresponding drop in the use of the DA B N2-FFdG pathway. As the basal SOS response in the dnaQ(«Q) strain was + αεθ αεLθ comparable to that in the dnaQ strain (SI Appendix,Fig.S5D), we ruled out the possibility that the increase in Pol IV-mediated 625 625 39 156 39 156 [Pol IV] (nM) TLS in the dnaQ(«Q*) strain resulted from elevation of Pol IV - - 78 78 312 312 expression levels. As the dnaQ(«Q*) mutation also reduces the processivity of n > 6 1.4 proofreading (27, 38), we examined if the increase in TLS in αεθ « 3rd 1.2 dnaQ( Q*) background was primarily due to a decrease in αεLθ 2nd 1.0 proofreading activity. To test this possibility, we abolished the TLS 0.8 exonuclease activity of the e subunit with the mutD5 mutation 1st 0.6 (SI Appendix,TableS1A) (50, 51) and assessed the fraction of 0.4 DA and TLS events over the G-BP(-) lesion. In the mutD5 0.2 ∼ ∼ Relative repriming 0.0 background, participation of TLS increased from 20 to 34%

0 but still remained significantly below the 55% value seen in the

200 400 600 800 dnaQ(« *) strain (Fig. 5A). Therefore, the additional increase [Pol IV] (nM) Q inTLSseenuponweakeningthee–cleft contact supports the Repriming model that the e subunit plays a gatekeeping role in regulating access of Pol IV during TLS in living cells.

e E. coli C Initiation Pol IV Quenching The Subunit Acts as a Gatekeeper for all 3 TLS Polymerases. t=0 t=30 or 270 s t=15~720 s Next, we asked whether the e–cleft interaction plays a similar gatekeeping role in Pol V-mediated TLS across TT-cyclobutane + N2-FFdG pyrimidine dimer (CPD) and TT(6–4) lesions. In the dnaQ No Pol IV Pol IV (30 s) Pol IV (270 s) background, replisomes stalled at TT-CPD and TT(6–4) were fully processed by the DA pathway as no Pol V-mediated TLS was 30 30 150 470 150 470 30 150 470 Time (s) measured. This lack of TLS is likely due to the absence of the 15 75 270 720 15 75 270 720 15 75 270 720 active form of Pol V (UmuD′2C) as UmuD is not processed into UmuD′ under non-SOS–induced conditions (Fig. 5B) (52). To n > 6 1.2 No Pol IV observe Pol V-mediated TLS, we engineered a strain that carries 3rd 1.0 Pol IV (30 sec) ′ ′ Pol IV (270 sec) umuD instead of umuD at the native umuDC locus (umuD C 2nd 0.8 strain); this strain constitutively expresses the active form of Pol V

st 0.6 (SI Appendix,TableS1B) (53). In this background, Pol V-mediated 1 ∼ ∼ – 0.4 TLS corresponded to 20% and 5% for TT-CPD and TT(6 4) lesions, respectively (Fig. 5B). Introduction of the dnaQ(«Q*) al- Repriming 0.2 Relative repriming lele in the umuD′C background further stimulated TLS to ∼70 and 0.0 ∼ – 0 50% for TT-CPD and TT(6 4), respectively (Fig. 5B). In con-

100 200 300 400 500 600 700 800 trast, introducing the proofreading-deficient mutD5 allele in the Time (s) umuD′C background, had no effect on the level of TLS, suggesting that the robust increase in TLS in the dnaQ(«Q*) strain resulted from an increased accessibility of Pol V to the clamp rather than Fig. 4. Pol IV diverts lesion-stalled replisomes from repriming to TLS. (A) from a defect in proofreading (Fig. 5B). Southern blot probes used in this study. A lesion-stalled replisome on the Finally, to investigate Pol II-mediated TLS, we introduced a leading-strand template reprimes downstream of the lesion, leaving an N2-acetylaminofluorene dG (G-AAF) lesion in the NarI sequence ssDNA gap between the lesion and a newly synthesized downstream context (SI Appendix, Supplemental Methods); this lesion can be primer. (B) Strengthening of the e–cleft interaction suppresses TLS at the bypassed by either Pol II- or Pol V-mediated TLS leading to −2or fork resulting in persistent repriming. (Left) Lesion-stalled replisomes error-free bypass, respectively (Fig. 5C) (5, 54). In contrast to what reprime downstream of N2-FFdG. Repriming and leading-strand replica- tion products resulting from replication of the N2-FFdG–containing tem- was observed for Pol IV and Pol V cognate lesions, Pol II- mediated TLS over the G-AAF lesion was comparably stimu- plate by wild-type (αeθ)oreL-containing (αeLθ) replisomes. Replication products were separated in a denaturing agarose gel and detected by lated in the dnaQ(«Q*) and mutD5 strains (Fig. 5D). The similar Southern blot with a mixture of leading-strand probes (900 and 1,901 nts) levels of TLS in the dnaQ(«Q*) and mutD5 backgrounds are most showninFig.4A.(Right) Integrated intensities of repriming replication likely related to the peculiar mechanism of Pol II-mediated −2 products (mean ± SD, n = 3). (C) Pol IV-mediated TLS at the fork kinetically competes with repriming. (Right, Upper) Replication of the N2-FFdG– containing template was initiated in the absence of Pol IV, and Pol IV (100 nM final) was added to the reaction 30 or 270 s after initiation. (Left) Fig. 4A.(Right, Lower) Quantitation of repriming products. Amounts of Reactions were quenched at the indicated time after initiation, and rep- repriming products were plotted relative to the amount of repriming lication products were separated in a denaturing agarose gel and de- products detected at the final time point (720 s) in the absence of Pol IV. The tected by Southern blot with a leading-strand probe (900 nts) shown in lines are linear connections of immediate time points (mean ± SD, n = 3).

6of11 | www.pnas.org/cgi/doi/10.1073/pnas.1914485116 Chang et al. Downloaded by guest on September 26, 2021 AB120 140 D120 DA DA DA TLS0 TLS-2 TLS TLS 100 120 100 100 80 80 80 60 60 60

Events (%) 40 Events (%) Events (%) 40 40

20 20 20

0 0 0 DA 86.6 40.4 62.7 DA 96.9 75.3 27.5 80.7103.4 90.7 45.2 87.2 DA 93.3 95.4 25.4 16.5 29.6 24.3 TLS 19.8 55.5 33.8 TLS 0.9 19.6 66.8 15.3 0.5 5.3 48.7 4.4 TLS0 0.5 0.1 0.2 20.3 0.2 3.0 + + + TLS-2 1.6 1.5 62.4 60.3 72.2 69.1 *) *) *) Q Q + Q *) *) ε ε Q Q ε dnaQ mutD5 umuD'C umuD'C dnaQ umuDC umuDC mutD5 + + dnaQ( ε o o dnaQ-frt G-BaP(-) dnaQ( ε umuD'C mutD5 umuD'C mutD5 + + umuD'C mutD5 o o + umuD'C dnaQ( umuD'C dnaQ( + + o umuD'C dnaQ( o o + o TT-CPD TT(6-4) G-AAF C Damage RecA AAF 3' CCGCGG 5' 3' CCA GTAC GG 5' Avoidance AAF 5' GG GCC 3' 5' GGT CATG CC 3' 3' CCGCGG 5' DA T T (DA) 5' GG 3' CA

Pol V BIOCHEMISTRY AAF Error-free 3' CCAGTACGG 5' AAF 3' CCGCGG 5' 3' CCGCGG 5' 5' GGTCATGCC 3' 3' CCGCGG 5' TLS Pathways 5' GGC 5' GGCGCC 3' 5' GGCGCC 3' Pol III (TLS0) TLS Stalled replication at AAF on AAF AAF Mutagenic the leading strand template with CG Pol II CG sequence heterology (green) on 3' C CGG 5' 3' C CGG 5' 3' CCGG 5' -2 Frameshift 5' G GCC 3' 5' GGCC 3' the replicated lagging strand 5' G GC TLS Pathways (TLS-2)

Fig. 5. Weakening the e–cleft interaction increases utilization of TLS in cells. (A) Extent of Pol IV-mediated TLS across G-BaP(-) (N2-(-)-transanti-benzo(a)pyrene dG) in indicated strains. (B) Extent of Pol V-mediated TLS across TT-CPD and TT(6–4) in indicated strains. (C) Lesion tolerance at a G-AAF adduct within the NarI site. Scheme of Pol II- or Pol V-mediated TLS across G-AAF at NarI recognition site. The 3 possible outcomes of lesion-tolerance pathways are illustrated: DA using homologous recombination results in copying the local sister chromatid sequence in green, TLS0 mediated by Pol V and TLS-2 (with −2 frameshift slippage intermediate) mediated by Pol II. (D)ExtentofPolII-andPolV-mediated TLS across G-AAF (N2-acetylaminofluorene dG) in indicated strains. To monitor Pol II-mediated TLS with −2 frameshift, the lesion-containing sequence was inserted into the genome with +2frameshiftsothatthelacZ+ sequence is synthesized only when −2frameshifttook place (see details in SI Appendix, Supplemental Methods). Results shown here are the average and the SD of at least 4 independent experiments.

frameshift mutagenesis, which involves elongation of a “slipped Discussion lesion terminus” (Fig. 5C) (54, 55). As revealed in the crystal Conformational Transitions of the Replicative Polymerase Underlie structure, Pol II is uniquely suited to elongate this frameshift in- Pathway Choice. During replication, replisomes encounter a di- termediate (56). Upon stalling at the G-AAF lesion, Pol III dis- verse set of challenges that can stall replicative DNA polymerases, sociates from the P/T junction, allowing for the formation of a −2 including DNA lesions and protein–DNA complexes (59, 60). It slippage intermediate; this intermediate is thermodynamically fa- remains poorly understood how replisomes select among distinct vored by the stacking energy provided by insertion of the aromatic rescue mechanisms to cope with these different situations. In this fluorene residue between the neighboring base pairs (57, 58). We study, we demonstrated that for replisomes stalled at leading- speculate that Pol II-mediated TLS was promoted in both back- strand lesions, the dynamic interaction between the exonuclease − grounds because in the mutD5 strain the formation of the 2 slip- subunit (e) of Pol III and the β2 clamp plays a crucial role in page intermediate is facilitated due to the lack of Pol III-dependent pathway choice between TLS at the fork and repriming (Fig. 6A). proofreading, while in the dnaQ(«Q*) strain, Pol II has increased We showed that the e–cleft interaction acts as a steric gate to limit access to the clamp, resulting in promoted extension of this slippage access of TLS polymerases to the P/T junction in a manner that is intermediate. largely independent of its exonuclease activity. This model is With respect to error-free bypass of G-AAF by Pol V, TLS is consistent with in vivo imaging demonstrating that Pol IV is only stimulated (∼7-fold) in the dnaQ(«Q*) strain compared with the weakly colocalized with the replisome in undamaged cells (14). mutD5 strain (Fig. 5D), consistent with the effect of the dnaQ(«Q*) Upon polymerase stalling at a lesion, Pol IV gains transient – on Pol V-mediated TLS across TT-CPD and TT(6 4) lesions. As access to the P/T junction by first binding to the cleft on the β2 weakening the e–cleft interaction in cells significantly increased the clampthatisoccupiedbythee subunit during processive rep- utilization of TLS over cognate lesions for all 3 TLS polymerases, lication (b through d in Fig. 6A). Weakening the e–cleft interac- we conclude that the e–cleft contact acts as a gatekeeper to reg- tion (“opening the gate,” transition from b to c in Fig. 6A) ulate access to the P/T junction. markedly increased TLS at the fork while only modestly decreasing

Chang et al. PNAS Latest Articles | 7of11 Downloaded by guest on September 26, 2021 A α Within stalled replisomes, recurring binding and unbinding Pol III core (αε) Template between the e subunit and the e–cleft form a kinetic barrier to ε Pol IV-clamp binding. Unbinding may be stimulated by a bulky A lesion on the leading lesion in the template strand; the inability of the narrow catalytic Pol IV β clamp 2 strand template cleft of the replicative polymerase to accommodate the lesion- containing template could promote opening of the Pol III–clamp complex. Alternatively, replicating Pol III core complexes may b) c) sample the open state (reversible transition between b and c in   Fig. 6A) regardless of the presence of blocking lesions (61). In Q L either case, strengthening the e–cleft interaction suppresses Processive these conformational transitions and therefore TLS, while replication weakening this interaction has the opposite effect.

a) d) Recent observations demonstrate that the E. coli replisome Closed states 1st Switching readily exchanges Pol III holoenzyme from cytosol during proc- essive replication (62–64), suggesting that Pol III core dissociation from the clamp may be required for Pol IV binding (61). In con- trast, our results suggest that the Pol III core remains bound to the

Open states Open β α– 2nd Switching 2 clamp during Pol IV-mediated TLS as strengthening the cleft interaction had little effect on TLS in vitro and in cells. This model f) e) Translesion is consistent with prior observations that both Pol III core and Pol synthesis IV can bind the clamp simultaneously, the toolbelt model (9, 65, 66). Single-molecule imaging of a minimal reconstitution of β2 clamp, Pol III core, and Pol IV demonstrated that higher con- centrations of the Pol III core and Pol IV promoted simultaneous binding of the clamp (61); this simultaneous binding could be fa- cilitated by interactions between polymerases and the replisome B that locally concentrate Pol III core and Pol IV. We propose that Replication block during exchange of the Pol III holoenzyme, another Pol III core Pol III rapidly binds the β2 clamp due to the preassociation of the Pol III Pol II holoenzyme with the replisome through its interaction with the Polymerase DnaB helicase (67, 68) (SI Appendix,Fig.S6A). In essence this is a Pol IV Repriming switching (1st) “dynamic toolbelt” model, in which rapid exchange of the Pol III Pol V core happens through the α–cleft while Pol IV carries out syn- thesis. Within this model the α–cleft interaction is maintained, Post-replicative HDGR TLS at the fork which is essential to prevent other factors from accessing the P/T TLS junction and compromising processive replication (7, 24). SOS It is also likely that the e–cleft interaction limits the size of the response TLS patch. Pol IV is expected to synthesize only a short patch Gap-filling Polymerase – synthesis switching (2nd) before it falls off the template and loses its CBM cleft interaction [Pol IV] (transitions from c through f in Fig. 6A) (9, 37). Following the dissociation of Pol IV from the e–cleft, the Pol III core must reestablish its e–cleft interaction before it can resume processive replication (transition from f to a in Fig. 6A). This step would be much faster than expected for diffusion-controlled binding of the Pol III core if Pol III remained bound to the β2 clamp while Pol IV performs TLS. Conversely, weakening the e–cleft interaction could result in longer TLS patches and slower replication due to rebinding Fig. 6. Conformational basis of pathway choice of lesion-stalled replisomes. (A) Conformational transitions of Pol III core complex during TLS. (a) Pol III core of Pol IV (Fig. 6A,transitionfromftodratherthantoa). (αeθ) occupies both α– and e–clefts during processive replication. (b and c) Upon encountering a lesion on the leading strand, Pol III core swings away from the Repriming Is a Failsafe Mechanism for Rescuing Stalled Replisomes.

P/T junction while remaining bound to the β2 clamp through the α-cleft in- We demonstrated that TLS and repriming compete kinetically to teraction. This conformational transition is either facilitated or suppressed by resolve stalled replisomes. Failure to carry out TLS at the fork in « « the dnaQ( Q) or the dnaQ( L) mutations, respectively. (d and e) Pol IV binds to a timely manner results in repriming, which acts as a failsafe to the e–cleft and take over the P/T junction and performs TLS past the lesion. (f) ensure replisome progression. A consequence of repriming is the After synthesizing a short patch, Pol IV swings away from the P/T junction and formation of an ssDNA gap behind the fork (SI Appendix, Fig. Pol III core reestablishes the e–cleft interaction and resumes processive repli- e e S6B). This gap is initially coated by an ssDNA binding protein cation. Within closed states, states a and b, the -cleft is occupied by the and subsequently converted to an RecA-ssDNA filament, which subunit. Within open states, states c though f, the e–cleft is not occupied by the e subunit. The θ subunit of Pol III core is not depicted for simplicity. (B)Pathway induces the SOS DNA damage response. Alternatively, TLS at choice for lesion-stalled replisomes during the SOS response: Repriming fol- the fork yields a continuous DNA product and therefore does lowed by gap-filling TLS vs. TLS at the fork. not lead to induction of the SOS response. Within our reconstitution, repriming downstream of an N2-FFdG adduct on the leading-strand template occurred quite slowly com- processive replication. Strengthening the e–cleft interaction paredwithPolIV-mediatedTLSatthe fork. Similar to prior ob- (“closing the gate,” transition from c to b in Fig. 6A) exerted the servations of repriming downstream of CPD lesions, we observed opposite effects. Therefore, the strength of this interaction is finely repriming occurring on the timescale of ∼4 min, implying that the tuned to maximize processive replication on undamaged DNA rate of repriming is likely independent of lesion identity (42, 43). while still enabling replisome remodeling by TLS polymerases While direct measurements of the rate of repriming in cells have upon the encounter of DNA damage. not been made, estimates (∼10 s) suggest that it could be much

8of11 | www.pnas.org/cgi/doi/10.1073/pnas.1914485116 Chang et al. Downloaded by guest on September 26, 2021 faster than in vitro observations (69, 70). Therefore, it is possible response (75). Even in the presence of highly elevated levels of that other factors present in the cellular environment, but not in Pol V, gap filling may still be the predominant mechanism as Pol current biochemical reconstitutions, may facilitate repriming. V activation requires a RecA-ssDNA filament downstream of On the other hand, the rate of TLS is controlled by at least 2 the stalled replisome (76–78). Weakening the e-gate still dra- factors: 1) the rate at which a TLS polymerase can bind the P/T matically increased Pol V-mediated TLS in cells (Fig. 5B), sug- junction and 2) the rate at which the TLS polymerase can insert gesting that the interaction between Pol III and the β2 clamp and extend past the lesion. As we have observed here, access of a likely influences polymerase switching independent of whether TLS polymerase to the P/T junction at the replication fork is TLS occurs at the fork or in a gap (Fig. 6B). Biochemical studies regulated, in part, by the dynamics of the e-gate. Increasing presented here and in prior work, demonstrate that Pol IV can concentrations of Pol IV led to more frequent TLS at the fork efficiently carry out TLS at the fork for at least a subset of lesions within our biochemical reconstitution. Similarly, when the level (79, 80). Cellular evidence for Pol IV-mediated TLS at the fork of Pol IV was constitutively elevated as a result of eliminating remains limited but the role of dinB in MMS-induced muta- LexA repressor binding sites in the promoter of the dinB gene genesis provides circumstantial evidence. 3meA is the major (dinBp-lexA-mut), the MMS-induced SOS response was decreased replication-blocking lesion created in MMS-treated cells and is relative to the wild-type strain, indicating that creation of ssDNA bypassed primarily by Pol IV in a largely error-free manner (81– gaps is suppressed (Fig. 6B and SI Appendix, Fig. S6 B and C). 84). Intriguingly, deletion of dinB increases MMS-induced mu- These results show that higher levels of Pol IV help to overcome tagenesis, which may result from more mutagenic TLS over the gatekeeping barrier of the e subunit by enabling Pol IV to 3meA by other polymerases at the fork (84, 85). However, we more readily capture the e open state. speculate that this increase is likely related to the creation of As cellular concentrations of all 3 TLS DNA polymerases are ssDNA gaps due to frequent repriming. In the absence of Pol IV, increased upon induction of the DNA damage-dependent SOS other polymerases mediate TLS over methyl lesions primarily in response, it is likely that TLS at the fork plays a more prominent a postreplicative manner because inefficient synthesis past the role in rescuing lesion stalled replisomes after SOS induction. lesion is likely outcompeted by repriming (Figs. 4B and 6B). As is During the early stages of the SOS response, when levels of TLS the case for untargeted mutagenesis by UV lesions (72), the polymerases are relatively low, the majority of lesion-stalled remainder of the gap can be filled in by mutagenic gap-filling replisomes are resolved by lesion skipping and most of the synthesis with Pol V being the major mutator. In contrast, MMS- resulting ssDNA gaps are repaired by DA (Fig. 6B). This is sup- induced mutagenesis is reduced by constitutive expression of Pol ported by our in vivo observations that only small fractions of IV (85). These observations are consistent with Pol IV-mediated lesion-stalled replisomes were resolved by TLS, which is likely TLS at the fork and suppression of ssDNA gaps (Fig. 6B). BIOCHEMISTRY through a gap-filling process (Fig. 5 A, B,andD). A potential Our result that the e-gate antagonizes TLS raises the question exception is Pol IV, which is present at relatively high levels even if other factors may stimulate TLS in cells and therefore con- in the SOS uninduced state and can efficiently bypass cognate tribute to mutagenesis. Intriguingly, we previously showed that Δ lesions (SI Appendix,Fig.S1I). In a dinB strain, replisomes stalled Pol IV is highly enriched near replisomes upon DNA damage in at Pol IV cognate DNA lesions would be predominantly resolved + a manner that was only partially dependent on interactions with by lesion skipping, leaving more ssDNA gaps than in a dinB strain. the β2 clamp (14), suggesting that additional interactions, pos- Indeed, when Pol IV-mediated TLS was completely abolished by sibly with replisome components, are required for localization of the deletion of dinB, the MMS-induced SOS response was highly Pol IV to stalled replisomes. Identifying these putative Pol IV– elevated compared with the wild-type strain (SI Appendix,Fig. replisome interactions, along with factors that may promote S6C), consistent with an increase in repriming. Collectively, path- repriming, will be important areas of future investigation. way choice is a net result of 2 counteracting molecular events—1) gatekeeping by the e subunit and 2) mass action of TLS poly- Materials and Methods — merases (71) with the latter changing throughout the SOS- Materials. Components of the E. coli replisome and other proteins were response. Failure of the TLS polymerase to gain access to the P/T expressed and purified as described in SI Appendix, Supplemental Materials. junction or the inability to quickly bypass the lesion upon binding E. coli strains were constructed by λ recombinase-mediated allelic exchange results in repriming and subsequent gap repair. and P1 transduction as described in SI Appendix, Supplemental Methods. Rolling-circle templates were constructed by inserting a lesion-containing or The Competition between TLS at the Fork and Repriming, and Its control oligomer at the EcoRI site into M13mp7(L2) phage genome as de- Impact on Mutagenesis. TLS at the fork prevents the creation of scribed in SI Appendix, Supplemental Methods. Lesion-containing plasmids ssDNA gaps by outcompeting repriming. This gap-suppressing for in vivo tolerance assay were constructed as previously described (46). effect of TLS at the fork may play a pivotal role in determining the extent of damage-induced mutagenesis. A recent study has Rolling-Circle DNA Replication. The E. coli replisome was assembled and stalled on the template in the presence of ATPγS (50 nM), dCTP, and dGTP demonstrated that damage-induced mutagenesis is not limited to (60 μM each). Replication reactions were initiated by adding dATP and dTTP the lesion site but instead can spread a few hundred nucleotides (60 μM each) together with [α−32P]dATP. Quenched reactions were run on a from the lesion (72). This extended patch of low-fidelity synthesis 0.6% alkaline agarose gel, and replication products were visualized by au- likely results from gap filling synthesis where bypass of the lesion toradiography. Details can be found in SI Appendix, Supplemental Methods. by the cognate TLS polymerase is followed by the sequential action of multiple polymerases, including the highly mutagenic Measurements of Damage-Induced SOS Responses. The GFP-based SOS- Pol V, to fill in the remaining ssDNA gap (Fig. 6B) (73, 74). response reporter strains were treated with DNA damaging agents for a However, if TLS occurs at the fork, error-prone TLS may be period and subsequently fixed with PBS containing 4% formaldehyde. Fixed limited to around the blocking lesion, as Pol III can readily regain cells were washed with and resuspended in PBS. GFP fluorescence from in- control of the P/T junction (Fig. 6B). dividual cells was measured by flow cytometry Within cells the extent to which TLS occurs at the fork versus in an ssDNA gap created by repriming remains unclear. Lesion Southern Blotting. Rolling-circle replication reactions were performed in the absence of [α−32P]dATP. Replication products were separated in a 0.6% identity, which plays a role in determining the rate of TLS, al- alkaline gel and transferred to a nylon membrane. Transferred DNA was most certainly affects the partitioning between these pathways. fixed to the membrane by UV illumination. To probe replication products, Lesions that are strongly blocking, such as UV-induced lesions, fixed membranes were incubated with 32P end-labeled oligomers, com- are likely to be predominantly resolved through repriming due to plementary to either a leading- or lagging-strand replication products and the absence of Pol V in the early stage of the UV-induced SOS unbound oligomers were removed by washing. Replication products were

Chang et al. PNAS Latest Articles | 9of11 Downloaded by guest on September 26, 2021 visualized by autoradiography. Details can be found in SI Appendix, All data are included in the main text and the SI Appendix. More details on Supplemental Methods. materials and methods can be found in the SI Appendix.

In Vivo Tolerance Assay and Sensitivity Assay. To assess partitioning of stalled ACKNOWLEDGMENTS. We thank Shingo Fujii (CNRS, Marseille), Daniel replisomes into either DA or TLS, a single replication blocking lesion was in- Semlow (Harvard Medical School), Johannes Walter (Harvard Medical corporated into the E. coli genome, as previously described (46). Subsequently School), and members of the J.J.L. laboratory for helpful discussions and lesion-incorporated cells were grown on X-gal–containing LB agar plates, and comments on the manuscript; and Deyu Li (University of Rhode Island) and the next day white and blue/white-sectored colonies, representing DA and TLS Yinsheng Wang (University of California, Riverside) for providing the N2- events, respectively, were counted. These colony counts were used to calculate FFdG– and N2-CEdG–containing oligomers, respectively. This work was sup- fractional DA and TLS as described in SI Appendix, Supplemental Methods.To ported by National Institutes of Health Grants R01 GM114065 (to J.J.L.) and examine sensitivity of strains to DNA-damaging agents, overnight cultures F32 GM113516 (to E.S.T.); Agence Nationale de la Recherche ForkRepair ANR were serially diluted and spotted onto NFZ- or MMS-containing LB-agar plates 11 BSV8 017 01 (to R.P.F.); and the Australian Research Council DP150100956 and incubated overnight. Dilution spots were pictured and analyzed. and DP180100858 (to N.E.D.).

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