Replication Fork Slowing and Reversal upon DNA Damage Require PCNA Polyubiquitination and ZRANB3 DNA Translocase Activity

Abstract

SummaryDNA damage tolerance during eukaryotic replication is orchestrated by PCNA ubiquitination. While monoubiquitination activates mutagenic translesion synthesis, polyubiquitination activates an error-free pathway, elusive in mammals, enabling damage bypass by template switching. Fork reversal is driven in vitro by multiple enzymes, including the DNA translocase ZRANB3, shown to bind polyubiquitinated PCNA. However, whether this interaction promotes fork remodeling and template switching in vivo was unknown. Here we show that damage-induced fork reversal in mammalian cells requires PCNA ubiquitination, UBC13, and K63-linked polyubiquitin chains, previously involved in error-free damage tolerance. Fork reversal in vivo also requires ZRANB3 translocase activity and its interaction with polyubiquitinated PCNA, pinpointing ZRANB3 as a key effector of error-free DNA damage tolerance. Mutations affecting fork reversal also induced unrestrained fork progression and chromosomal breakage, suggesting fork remodeling as a global fork slowing and protection mechanism. Targeting these fork protection systems represents a promising strategy to potentiate cancer chemotherapy.

Replication Fork Reversal: Players and Guardians

“…Indeed, SMARCAL1, ZRANB3, and HLTF recognize different types of fork structures in vitro , suggesting that cells might use different factors depending on the particular type of replication intermediate (Betous et al, 2013; Hishiki et al, 2015; Kile et al, 2015). Along the same line, the studies of Kolinjivadi et al (2017), Taglialatela et al (2017), and Vujanovic et al (2017) clearly show that SMARCAL1 or ZRANB3 depletion does not fully abrogate reversed fork formation, supporting the idea that fork reversal is not mediated by a single fork remodeler and that different structures might arise even when using the same type of replication challenge. Moreover, other DNA translocases, including RAD54 (Bugreev et al, 2011) and FANCM (Gari et al, 2008), can mediate fork reversal in vitro , suggesting that additional factors may contribute to this process.…”

“…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). PCNA post-translational modifications are triggered by the formation of RPA-coated ssDNA at stalled forks and are key regulators of pathway choice between error-prone translesion DNA synthesis and error-free template switching mechanisms (Mailand et al, 2013).…”

“…Interestingly, Kolinjivadi et al (2017), Tagliatela et al (2017), and Vujanovic et al (2017) show that fork reversal is not impaired under conditions that lead to formation of unstable RAD51 nucleofilaments and/or inefficient loading of RAD51 on ssDNA (Figure 1C), such as in BRCA1/2-mutant cells or in cells expressing a dominant RAD51 mutant allele, RAD51-T131P, which was recently reported to destabilize RAD51 nucleofilaments (Wang et al, 2015). The molecular steps that allow replication forks to reverse under conditions of inefficient RAD51 loading remain to be defined.…”

“…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).…”