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Postreplication repair (PRR) pathways play important roles in restarting stalled replication forks and regulating mutagenesis. In yeast, Rad5-mediated damage avoidance and Rad18-mediated translesion synthesis (TLS) are two forms of PRR. Two Rad5-related proteins, SHPRH and HLTF, have been identified in mammalian cells, but their specific roles in PRR are unclear. Here, we show that HLTF and SHPRH suppress mutagenesis in a damage-specific manner, preventing mutations induced by UV and MMS, respectively. Following UV, HLTF enhances PCNA monoubiquitination and recruitment of TLS polymerase η, while also inhibiting SHPRH function. In contrast, MMS promotes the degradation of HLTF and the interactions of SHPRH with Rad18 and polymerase κ. Our data suggest not only that cells differentially utilize HLTF and SHPRH for different forms of DNA damage, but also, surprisingly, that HLTF and SHPRH may coordinate the two main branches of PRR to choose the proper bypass mechanism for minimizing mutagenesis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The function of the ATR (ataxia-telangiectasia mutated and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is central to the cellular response to replication stress and DNA damage. In order to better understand the function of this complex, we have studied its interaction with DNA. We find that both ATR and ATRIP associate with chromatin in vivo, and they exist as a large molecular weight complex that can bind single-stranded (ss)DNA cellulose in vitro. Although replication protein A (RPA) is sufficient for the recruitment of ATRIP to ssDNA, we show that a distinct ATR-ATRIP complex is able to bind to DNA with lower affinity in the absence of RPA. In this latter complex, we show that neither ATR nor ATRIP are able to bind DNA individually, nor do they bind DNA in a cooperative manner. However, the addition of HeLa nuclear extract is able to reconstitute the DNA binding of both ATR and ATRIP, suggesting the requirement for an additional protein activity. We also show that ATR is necessary for ATRIP to bind DNA in this low affinity mode and to form a large DNA binding complex. These observations suggest that there are at least two in vitro ATR-ATRIP DNA binding complexes, one which binds DNA with high affinity in an RPA-dependent manner and a second, which binds DNA with lower affinity in an RPA-independent manner but which requires an as of yet unidentified protein.

Using the Xenopus egg extract system, we investigated the involvement of DNA replication in activation of the DNA damage checkpoint. We show here that DNA damage slows replication in a checkpoint-independent manner and is accompanied by replication-dependent recruitment of ATR and Rad1 to chromatin. We also find that the replication proteins RPA and Polalpha accumulate on chromatin following DNA damage. Finally, damage-induced Chk1 phosphorylation and checkpoint arrest are abrogated when replication is inhibited. These data indicate that replication is required for activation of the DNA damage checkpoint and suggest a unifying model for ATR activation by diverse lesions during S phase.