Replication Fork Reversal: Players and Guardians

Abstract

Replication fork reversal is a rapidly emerging and remarkably frequent mechanism of fork stabilization in response to genotoxic insults. Here, we summarize recent findings that uncover key molecular determinants for reversed fork formation and describe how the homologous recombination factors BRCA1, BRCA2, and RAD51 protect these structures from extended nucleolytic degradation.

Keywords: BRCA1; BRCA2; DNA repair; DNA replication; fork regression; genome instability; homologous recombination; replication fork restart; replication fork reversal; replication stress.

Figure 1. Mechanisms of reversed replication fork formation

(A) Accumulation of ssDNA at uncoupled replication forks coated by RPA promotes the recruitment of the E2–E3 ubiquitin conjugating enzymes, which mediate PCNA monoubiquitination at lysine 164 (K164), as well as UBC13-dependent K63-linked polyubiquitination of the same residue. Polyubiquitinylated PCNA interacts with the ZRANB3 translocase, which promotes replication fork reversal. (B) BRCA2 promotes RAD51 binding to replicating DNA. RAD51 directly interacts with Pol α to facilitate DNA synthesis and avoid gap formation. Persistent ssDNA gaps are remodeled into a reversed replication fork by the translocase activity of SMARCAL1. (C) Unstable RAD51 nucleofilaments and/or inefficient loading of RAD51 on ssDNA, such as in BRCA2-deficient or RAD51-T131P cells, does not impair fork reversal, either because fork reversal does not require extensive homology search at a distance or because the limited loading of RAD51 is sufficient to recruit specific translocases on the uncoupled fork.

Figure 2. Mechanisms of reversed replication fork protection and restart

(A) BRCA2 stabilizes the RAD51 nucleofilament on the regressed arms, thereby preventing MRE11, CtIP and EXO1-dependent resection of the regressed arms. RECQ1 promotes restart of the reversed replication forks. DNA2/WRN also promote reversed fork restart through a mechanism that entails limited DNA2-dependent processing of the regressed arm. (B) Reversed forks are extensively degraded by MRE11, CtIP and EXO1 in BRCA2-deficient cells. PTIP, MLL3/4 and RAD52 promote MRE11 recruitment on the stalled forks. The initial limited degradation of regressed arm generates a reversed fork with a 3′ ssDNA flap that is a substrate for MUS81 cleavage. MUS81 cleavage produces a migrating bubble that promotes POLD3-dependent DNA synthesis. If MUS81 does not promptly cleave the partially resected forks, the nucleolytic degradation might quickly proceed to degrade nascent strands behind the junction, finally leading to extensively resected forks. After extended fork resection, reannealing of the parental strands might lead to “backtracking” of the fork. As a consequence of fork backtracking, a new reversal event may occur, possibly promoting a new MUS81 cleavage event.