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).