Replication stalling activates SSB for recruitment of DNA damage tolerance factors

Abstract

Translesion synthesis (TLS) polymerases bypass DNA lesions that block replicative polymerases, allowing cells to tolerate DNA damage encountered during replication. It is well known that most bacterial TLS polymerases must interact with the sliding-clamp processivity factor to carry out TLS, but recent work in Escherichia coli has revealed that single-stranded DNA-binding protein (SSB) plays a key role in enriching the TLS polymerase Pol IV at stalled replication forks in the presence of DNA damage. It remains unclear how this interaction with SSB enriches Pol IV in a stalling-dependent manner given that SSB is always present at the replication fork. In this study, we use single-molecule imaging in live E. coli cells to investigate this SSB-dependent enrichment of Pol IV. We find that Pol IV is enriched through its interaction with SSB in response to a range of different replication stresses and that changes in SSB dynamics at stalled forks may explain this conditional Pol IV enrichment. Finally, we show that other SSB-interacting proteins are likewise selectively enriched in response to replication perturbations, suggesting that this mechanism is likely a general one for enrichment of repair factors near stalled replication forks.

“The Translesion Polymerase Pol Y1 is a Constitutive Component of theB. subtilisReplication Machinery”

“…In E. coli , changes in the localization and dynamics of TLS polymerases have been observed upon DNA damage or other perturbations to replication; in particular, Pol IV is strongly recruited to sites of replication upon induction of DNA damage by methyl methanesulfonate (MMS) or ultraviolet (UV) light (Figure 6, top right panel). (15) In this study, we characterized the localization and dynamics of Pol Y1 after different treatments with the drug 4-NQO; consistent with previous work,(25) we found that Pol Y1, but not Pol Y2, mediates cell survival to 4-NQO treatment. Surprisingly, we observed little or no change in Pol Y1 behavior, including its colocalization with sites of replication and its dynamics, upon 4-NQO treatment (Figure 6, bottom right panel).…”

“…In E. coli , the interaction with the clamp is essential for Pol IV activity,(56, 54, 14) but the interaction with SSB plays a greater role in Pol IV enrichment near sites of replication. (13, 15) We constructed C-terminal HaloTag fusions to both CBM mutants, in combination with the DnaX-mYPet replisome marker, and imaged cells under the same conditions as for WT Pol Y1. Radial distribution function analysis revealed a complete loss of Pol Y1-DnaX colocalization for both CBM mutants ( g ( r ) ≈ 0.157 and 0.0938 at short r values for Pol Y1-CBM-mut1 and Pol Y1-CBM-mut2, respectively) (Figure 4B).…”

“…In E. coli , the Pol Y1 homolog Pol IV is recruited to sites of replication in response to a variety of replication perturbations, including treatment with cognate DNA damaging agents, which generate DNA lesions that Pol IV can bypass efficiently. (14, 15) Given that Pol Y1 colocalizes with sites of replication during normal growth, we next asked whether this enrichment would increase upon DNA damage. To address this question, we chose to investigate the response of Pol Y1 to treatment with the drug 4-NQO; as shown previously, Pol Y1 promotes cell survival upon 4-NQO treatment.…”

“…(18, 21) Although SOS induction of Pol IV plays an important role in its response to DNA damage, selective recruitment of Pol IV to the replication fork upon replication stalling was observed in a constitutively SOS-induced strain where Pol IV levels were unchanged upon DNA damage. (14, 15) Thus, the lack of SOS induction of Pol Y1 is likely not sufficient to explain the lack of response to 4-NQO treatment. These results raise the possibility that Pol Y1 promotes cell survival upon DNA damage through different mechanisms than Pol IV.…”

“…For trajectories containing exactly four steps, the following analytical expression describes the probability distribution: where A n is the fraction of molecules in the n th species and D n represents the diffusion coefficient of that species. (15, 40) Fitting was performed subject to the normalization constraint: Longer trajectories were truncated to retain only the first four steps before use in this analysis.…”