Replication stalling activates SSB for recruitment of DNA damage tolerance factors.

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.

Compartmentalization of the replication fork by single-stranded DNA-binding protein regulates translesion synthesis.

Processivity clamps tether DNA polymerases to DNA, allowing their access to the primer-template junction. In addition to DNA replication, DNA polymerases also participate in various genome maintenance activities, including translesion synthesis (TLS). However, owing to the error-prone nature of TLS polymerases, their association with clamps must be tightly regulated. Here we show that fork-associated ssDNA-binding protein (SSB) selectively enriches the bacterial TLS polymerase Pol IV at stalled replication forks. This enrichment enables Pol IV to associate with the processivity clamp and is required for TLS on both the leading and lagging strands. In contrast, clamp-interacting proteins (CLIPs) lacking SSB binding are spatially segregated from the replication fork, minimally interfering with Pol IV-mediated TLS. We propose that stalling-dependent structural changes within clusters of fork-associated SSB establish hierarchical access to the processivity clamp. This mechanism prioritizes a subset of CLIPs with SSB-binding activity and facilitates their exchange at the replication fork.

XLF acts as a flexible connector during non-homologous end joining.

Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double-strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here, we use Xenopus laevis egg extract to investigate the role of the intrinsically disordered C-terminal tail of the XRCC4-like factor (XLF), a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku-binding motif (KBM) at the extreme C-terminus are required for end joining. Although the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe end synapsis in real-time show that this defect is due to a failure to closely align DNA ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku, while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.

The Role of Noncognate Sites in the 1D Search Mechanism of EcoRI.

A one-dimensional (1D) search is an essential step in DNA target recognition. Theoretical studies have suggested that the sequence dependence of 1D diffusion can help resolve the competing demands of a fast search and high target affinity, a conflict known as the speed-selectivity paradox. The resolution requires that the diffusion energy landscape is correlated with the underlying specific binding energies. In this work, we report observations of a 1D search by quantum dot-labeled EcoRI. Our data supports the view that proteins search DNA via rotation-coupled sliding over a corrugated energy landscape. We observed that whereas EcoRI primarily slides along DNA at low salt concentrations, at higher concentrations, its diffusion is a combination of sliding and hopping. We also observed long-lived pauses at genomic star sites, which differ by a single nucleotide from the target sequence. To reconcile these observations with prior biochemical and structural data, we propose a model of search in which the protein slides over a sequence-independent energy landscape during fast search but rapidly interconverts with a "hemispecific" binding mode in which a half site is probed. This half site interaction stabilizes the transition to a fully specific mode of binding, which can then lead to target recognition.