Replication-Coupled DNA Repair.

Complete and accurate DNA replication requires the progression of replication forks through DNA damage, actively transcribed regions, structured DNA and compact chromatin Recent studies have revealed a remarkable plasticity of the replication process in dealing with these obstacles, which includes modulation of replication origin firing, of the architecture of replication forks, and of the functional organization of the replication machinery in response to replication stress However, these specialized mechanisms also expose cells to potentially dangerous transactions while replicating DNA In this Review, we discuss how replication forks are actively stalled, remodelled, processed, protected and restarted in response to specific types of stress We also discuss adaptations of the replication machinery and the role of chromatin modifications during these transactions Finally, we discuss interesting recent data on the relevance of replication fork plasticity to human health, covering its role in tumorigenesis, its crosstalk with innate immunity responses and its potential as an effective cancer therapy target

The plasticity of DNA replication forks in response to clinically relevant genotoxic stress

The replisome, the molecular machine dedicated to copying DNA, encounters a variety of obstacles during S phase. Without a proper response to this replication stress, the genome becomes unstable, leading to disease, including cancer. The immediate response is localized to the stalled replisome and includes protection of the nascent DNA. A number of recent studies have provided insight into the factors recruited to and responsible for protecting stalled replication forks. In response to replication stress, the SNF2 family of DNA translocases has emerged as being responsible for remodeling replication forks in vivo. The protection of stalled replication forks requires the cooperation of RAD51, BRCA1, BRCA2, and many other DNA damage response proteins. In the absence of these fork protection factors, fork remodeling renders them vulnerable to degradation by nucleases and helicases, ultimately compromising genome integrity. In this review, we focus on the recent progress in understanding the protection, processing, and remodeling of stalled replication forks in mammalian cells.

/pdf/advances-in-understanding-dna-processing-and-protection-at-2l7uj63l7k.pdf

Advances in understanding DNA processing and protection at stalled replication forks.

Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency

Homologous recombination (HR) and Fanconi Anemia (FA) pathway proteins in addition to their DNA repair functions, limit nuclease-mediated processing of stalled replication forks. However, the mechanism by which replication fork degradation results in genome instability is poorly understood. Here, we identify RIF1, a non-homologous end joining (NHEJ) factor, to be enriched at stalled replication forks. Rif1 knockout cells are proficient for recombination, but displayed degradation of reversed forks, which depends on DNA2 nuclease activity. Notably, RIF1-mediated protection of replication forks is independent of its function in NHEJ, but depends on its interaction with Protein Phosphatase 1. RIF1 deficiency delays fork restart and results in exposure of under-replicated DNA, which is the precursor of subsequent genomic instability. Our data implicate RIF1 to be an essential factor for replication fork protection, and uncover the mechanisms by which unprotected DNA replication forks can lead to genome instability in recombination-proficient conditions.

/pdf/rif1-promotes-replication-fork-protection-and-efficient-2zllft64k3.pdf

RIF1 promotes replication fork protection and efficient restart to maintain genome stability.

https://hal.archives-ouvertes.fr/hal-02144125/document

RADX Modulates RAD51 Activity to Control Replication Fork Protection.

Fork reversal is a common response to replication stress, but it generates a DNA end that is susceptible to degradation. Many fork protection factors block degradation, but how they work remains unclear. Here, we find that 53BP1 protects forks from DNA2-mediated degradation in a cell type–specific manner. Fork protection by 53BP1 reduces S-phase DNA damage and hypersensitivity to replication stress. Unlike BRCA2, FANCD2, and ABRO1 that protect reversed forks generated by SMARCAL1, ZRANB3, and HLTF, 53BP1 protects forks remodeled by FBH1. This property is shared by the fork protection factors FANCA, FANCC, FANCG, BOD1L, and VHL. RAD51 is required to generate the resection substrate in all cases. Unexpectedly, BRCA2 is also required for fork degradation in the FBH1 pathway or when RAD51 activity is partially compromised. We conclude that there are multiple fork protection mechanisms that operate downstream of at least two RAD51-dependent fork remodeling pathways.

https://advances.sciencemag.org/content/advances/6/46/eabc3598.full.pdf

Two replication fork remodeling pathways generate nuclease substrates for distinct fork protection factors

Replication-coupled DNA repair and damage tolerance mechanisms overcome replication stress challenges and complete DNA synthesis. These pathways include fork reversal, translesion synthesis, and repriming by specialized polymerases such as PRIMPOL. Here, we investigated how these pathways are used and regulated in response to varying replication stresses. Blocking lagging-strand priming using a POLα inhibitor slows both leading- and lagging-strand synthesis due in part to RAD51-, HLTF-, and ZRANB3-mediated, but SMARCAL1-independent, fork reversal. ATR is activated, but CHK1 signaling is dampened compared to stalling both the leading and lagging strands with hydroxyurea. Increasing CHK1 activation by overexpressing CLASPIN in POLα-inhibited cells promotes replication elongation through PRIMPOL-dependent repriming. CHK1 phosphorylates PRIMPOL to promote repriming irrespective of the type of replication stress, and this phosphorylation is important for cellular resistance to DNA damage. However, PRIMPOL activation comes at the expense of single-strand gap formation, and constitutive PRIMPOL activity results in reduced cell fitness.

Archana Krishnamoorthy

Papers

RADX Modulates RAD51 Activity to Control Replication Fork Protection.

Two replication fork remodeling pathways generate nuclease substrates for distinct fork protection factors

CHK1 phosphorylates PRIMPOL to promote replication stress tolerance

RADX prevents genome instability by confining replication fork reversal to stalled forks.