Smarcal1-Mediated Fork Reversal Triggers Mre11-Dependent Degradation of Nascent DNA in the Absence of Brca2 and Stable Rad51 Nucleofilaments

Abstract

SummaryBrca2 deficiency causes Mre11-dependent degradation of nascent DNA at stalled forks, leading to cell lethality. To understand the molecular mechanisms underlying this process, we isolated Xenopus laevis Brca2. We demonstrated that Brca2 protein prevents single-stranded DNA gap accumulation at replication fork junctions and behind them by promoting Rad51 binding to replicating DNA. Without Brca2, forks with persistent gaps are converted by Smarcal1 into reversed forks, triggering extensive Mre11-dependent nascent DNA degradation. Stable Rad51 nucleofilaments, but not RPA or Rad51T131P mutant proteins, directly prevent Mre11-dependent DNA degradation. Mre11 inhibition instead promotes reversed fork accumulation in the absence of Brca2. Rad51 directly interacts with the Pol α N-terminal domain, promoting Pol α and δ binding to stalled replication forks. This interaction likely promotes replication fork restart and gap avoidance. These results indicate that Brca2 and Rad51 prevent formation of abnormal DNA replication intermediates, whose processing by Smarcal1 and Mre11 predisposes to genome instability.

Replication Fork Reversal: Players and Guardians

“…In particular, Kolinjivadi et al (2017) propose that SMARCAL1 remodels forks with persistent ssDNA gaps at the fork junction into reversed fork structures and Taglialatela et al (2017) suggest that the RPA-binding activity of SMARCAL1 is required for its replication function, in agreement with previous biochemical studies (Betous et al, 2013). At the same time, Vujanovic et al (2017) show that ZRANB3 interacts with polyubiquitinated PCNA to promote fork remodeling (Figure 1A).…”

“…However, the molecular determinants required to protect the integrity of regressed arms until forks are restarted are unknown. Here, we review recent articles that provide a fresh perspective on these important issues by defining a key function of two translocases of the SWI/SNF protein family, i.e., ZRANB3 and SMARCAL1, in reversed fork formation (Kolinjivadi et al, 2017; Taglialatela et al, 2017; Vujanovic et al, 2017) and a key function of the breast cancer susceptibility proteins BRCA1 and BRCA2 in reversed fork protection (Kolinjivadi et al, 2017; Lemacon et al, 2017; Mijic et al, 2017; Taglialatela et al, 2017). …”

“…Using an elegant combination of single-molecule DNA fiber and electron microscopy approaches, Kolinjivadi et al (2017), Taglialatela et al (2017) and Vujanovic et al (2017) demonstrate that the translocase activities of SMARCAL1 and ZRANB3 are required for replication fork reversal. In particular, Kolinjivadi et al (2017) propose that SMARCAL1 remodels forks with persistent ssDNA gaps at the fork junction into reversed fork structures and Taglialatela et al (2017) suggest that the RPA-binding activity of SMARCAL1 is required for its replication function, in agreement with previous biochemical studies (Betous et al, 2013).…”

The MRE11–RAD50–NBS1 Complex Conducts the Orchestration of Damage Signaling and Outcomes to Stress in DNA Replication and Repair

“…Thus, other proteins help regulate MRN-mediated nascent DNA degradation (147–150). The Fanconi anemia and breast cancer protein BRCA2 blocks MRE11-mediated resection of nascent ssDNA by promoting formation of stable nucleofilaments of RAD51 on nascent DNA (147, 151). However, cyclin-dependent kinase 2 (CDK2)–dependent phosphorylation of the BRCA2 C terminus results in disassembly of RAD51 filaments (152).…”

Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

“…Therefore, it is likely that ATP binding and hydrolysis play a significant role in the conformation and stability of hRAD51 NPFs. Understanding the disassembly of hRAD51 from ssDNA is important as the intrinsic stability of hRAD51‐ssDNA NPFs is likely to affect the reaction of strand exchange during homologous recombination (Taylor et al , 2015, 2016) and the ability of hRAD51 to protect ssDNA gaps present at stalled replication forks (Kolinjivadi et al , 2017). Yet, the mechanism of hRAD51 disassembly from ssDNA and how it is affected by factors such as ssDNA template tension and ATP hydrolysis remain unknown.…”