Persistently stalled replication forks inhibit nucleotide excision repair in trans by sequestering Replication protein A

Abstract

Rev3, the catalytic subunit of DNA polymerase ζ, is essential for translesion synthesis of cytotoxic DNA photolesions, whereas the Rev1 protein plays a noncatalytic role in translesion synthesis. Here, we reveal that mammalian Rev3−/− and Rev1−/− cell lines additionally display a nucleotide excision repair (NER) defect, specifically during S phase. This defect is correlated with the normal recruitment but protracted persistence at DNA damage sites of factors involved in an early stage of NER, while repair synthesis is affected. Remarkably, the NER defect becomes apparent only at 2 h post-irradiation indicating that Rev3 affects repair synthesis only indirectly, rather than performing an enzymatic role in NER. We provide evidence that the NER defect is caused by scarceness of Replication protein A (Rpa) available to NER, resulting from its sequestration at stalled replication forks. Also the induction of replicative stress using hydroxyurea precludes the accumulation of Rpa at photolesion sites, both in Rev3−/− and in wild-type cells. These data support a model in which the limited Rpa pool coordinates replicative stress and NER, resulting in increased cytotoxicity of ultraviolet light when replicative stress exceeds a threshold.

Mutations in Replicative Stress Response Pathways Are Associated with S Phase-specific Defects in Nucleotide Excision Repair

“…Another recent study of particular relevance here showed that Rev3 Ϫ/Ϫ murine embryonic fibroblasts, which lack DNA polymerase activity required for efficient bypass of UV-induced 6-4PPs, exhibit NER deficiency specifically during S phase (17). Rev3 Ϫ/Ϫ murine embryonic fibroblasts (which, like pol-deficient counterparts, are ostensibly characterized by enhanced replicative stress) also exhibited marked reduction in the recruitment of RPA to UV-irradiated nuclear regions.…”

“…Partial Depletion of Rfa1 Inhibits SPR-We originally speculated (15), and more recently others have presented evidence supporting the notion (17), that abnormal accumulation of RPA1-3 at damaged DNA replication forks could reduce the availability of this complex for NER during S phase. To directly test this model in yeast, we investigated the effect of partial RPA depletion on CPD removal in replicating cells.…”

“…However, one potential connection relates to the fact that cells derived from pol-deficient skin cancer-prone xeroderma pigmentosum variant (XPV) patients or from ATR-deficient Seckel syndrome patients each exhibit increased replication fork stalling in response to UV (18,19). Based on this, we (15) and others (17) have postulated a functional link between levels of replicative stress and NER efficiency.…”

Roles of mutagenic translesion synthesis in mammalian genome stability, health and disease

“…Also surprisingly, both Rev3 and Rev1 deficiency result in a concomitant defect of nucleotide excision repair during S phase. This defect results from the sequestration of RPA to single-strand DNA at arrested replication forks in the Rev1 KO and Rev3 KO cells, precluding RPA to exert its role in coupling early and late steps in nucleotide excision repair [80]. The positive feedback loop between translesion synthesis defects and nucleotide excision repair defects may further exacerbate the induction of genomic instability by DNA damage.…”

Replication protein A prevents promiscuous annealing between short sequence homologies: Implications for genome integrity

“…Alternatively, the stalled replication forks resulting from replication stress, or forks remodeled by DNA translocases to form structures resembling Holliday junctions, could be acted on by the Mus81‐Mms4/EME1 and/or Slx1‐Slx4 endonucleases resulting in DSBs . The pool of free RPA also becomes limiting when mouse embryonic fibroblasts deficient for DNA polymerase ζ are treated with UV light due to sequestration of RPA at damaged replication forks, and this interferes with global nucleotide excision repair .…”