PIP domain ͉ UBZ domain ͉ PCNA ubiquitination ͉ exchange

The Y family DNA polymerase Rev1 has been proposed to play a regulatory role in the replication of damaged templates. To elucidate the mechanism by which Rev1 promotes DNA damage bypass, we have analyzed the progression of replication on UV light-damaged DNA in mouse embryonic fibroblasts that contain a defined deletion in the N-terminal BRCT domain of Rev1 or that are deficient for Rev1. We provide evidence that Rev1 plays a coordinating role in two modes of DNA damage bypass, i.e., an early and a late pathway. The cells carrying the deletion in the BRCT domain are deficient for the early pathway, reflecting a role of the BRCT domain of Rev1 in mutagenic translesion synthesis. Rev1-deficient cells display a defect in both modes of DNA damage bypass. Despite the persistent defect in the late replicational bypass of fork-blocking (6-4)pyrimidine-pyrimidone photoproducts, overall replication is not strongly affected by Rev1 deficiency. This results in almost completely replicated templates that contain gaps encompassing the photoproducts. These gaps are inducers of DNA damage signaling leading to an irreversible G 2 arrest. Our results corroborate a model in which Rev1-mediated DNA damage bypass at postreplicative gaps quenches irreversible DNA damage responses.Unrepaired DNA damage usually leads to an arrest of replicative polymerases. Nonetheless, prokaryotic and eukaryotic cells display progression of replication on damaged templates, allowing replication to be completed and averting replication fork collapse (12). Direct bypass of the fork-blocking lesion, permitting replication to proceed with little delay, would be an attractive mechanism to release an arrested replicon. Several early studies using bacteria and mammalian cells, however, have indicated that replicating cells exposed to UV light initially synthesize smaller DNA strands than undamaged cells (reviewed in reference 32). At a later stage, these molecules are converted into DNA of high molecular weight, possibly via late, postreplicative, filling of the lesion-containing gap by socalled postreplication repair. The presence of single-stranded gaps in both sister chromatids behind a replication fork was recently visualized by electron microscopy on DNA of UVexposed budding yeast Saccharomyces cerevisiae (34).Damage avoidance and translesion synthesis are two pathways that allow cells to replicate damaged templates (3, 12). Damage avoidance (also called template switching-dependent synthesis) uses the undamaged sister chromatid as a template. This can be achieved by replication fork regression or by strand invasion with the sister chromatid and results in error-free bypass of DNA lesions (7, 65). Translesion synthesis, on the other hand, is characterized by insertion of a nucleotide opposite the lesion by specialized DNA polymerases of the Y family (48). The reduced stringency of the active site and a lack of proofreading activity of translesion synthesis polymerases imply that translesion synthesis is an inherently mutagenic process.The Y family poly...

Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteins retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. In more general terms, we suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal.