Activation of Ubiquitin-Dependent DNA Damage Bypass Is Mediated by Replication Protein A

Abstract

SummaryReplicative DNA damage bypass, mediated by the ubiquitylation of the sliding clamp protein PCNA, facilitates the survival of a cell in the presence of genotoxic agents, but it can also promote genomic instability by damage-induced mutagenesis. We show here that PCNA ubiquitylation in budding yeast is activated independently of the replication-dependent S phase checkpoint but by similar conditions involving the accumulation of single-stranded DNA at stalled replication intermediates. The ssDNA-binding replication protein A (RPA), an essential complex involved in most DNA transactions, is required for damage-induced PCNA ubiquitylation. We found that RPA directly interacts with the ubiquitin ligase responsible for the modification of PCNA, Rad18, both in yeast and in mammalian cells. Association of the ligase with chromatin is detected where RPA is most abundant, and purified RPA can recruit Rad18 to ssDNA in vitro. Our results therefore implicate the RPA complex in the activation of DNA damage tolerance.

BRCA1promotes the ubiquitination of PCNA and recruitment of translesion polymerases in response to replication blockade

“…The binding of RPA to ssDNA is thought to be an earlier event during the activation of DDT (12,18). Likely through a direct protein-protein interaction, RPA70 recruits Rad18 to ssDNA associated with replication fork stalling, where Rad18 then initiates the ubiquitination of PCNA and the DDT pathway.…”

“…For example, TLS polymerase eta (Polη) can bypass a thymidine dimer induced by UV with high fidelity (16,17). In addition to the enzymes involved in ubiquitinating PCNA, the single-stranded DNA (ssDNA) binding protein, replication protein A (RPA), is also important for the ubiquitination of PCNA and functions to recruit the E3 ligase, Rad18 (12,18). Both branches of the DDT pathways appear to be more complex in mammals.…”

Cooperation of replication Protein A with the ubiquitin ligase Rad18 in DNA damage bypass

“…14 Moreover, the use of a temperature-sensitive mutant of an essential kinase gene responsible for DNA replication initiation, cdc7 ts , allowed us to demonstrate that lack of the modification outside of S phase is due to the absence of active replication forks rather than to a particular cell cycle control mechanism. This observation confirmed findings in Xenopus laevis egg extracts, where ubiquitylation of PCNA was also shown to depend on active replication.…”

“…6,13 Until recently, the upstream signals that initiate PCNA ubiquitylation were not well defined. We have now analyzed the requirements for this modification in budding yeast 14 and found in vivo and in vitro evidence for an involvement of the ssDNA-binding RPA complex in the activation of DNA damage tolerance.…”

TopBP1-mediated DNA processing during mitosis

“…54 Rather RPA-bound ssDNA generated by uncoupling of the replication fork and the replicative helicases recruits Rad18 to stalled forks. 54,55 Alternatively, TLS polymerases may be recruited to the stalled fork by the Rev1 scaffold protein. 56 In DT40 cells, Rev1 and PCNA ubiquitylation at K164 are central to TLS-mediated restart or gap filling, respectively.…”