REV1 protein is a eukaryotic member of the Y family of DNA polymerases involved in the tolerance of DNA damage by replicative bypass. The precise role(s) of REV1 in this process is not known. Here we show, by using the yeast two-hybrid assay and the glutathione S-transferase pull-down assay, that mouse REV1 can physically interact with ubiquitin. The association of REV1 with ubiquitin requires the ubiquitin-binding motifs (UBMs) located at the C terminus of REV1. The UBMs also mediate the enhanced association between monoubiquitylated PCNA and REV1. In cells exposed to UV radiation, the association of REV1 with replication foci is dependent on functional UBMs. The UBMs of REV1 are shown to contribute to DNA damage tolerance and damage-induced mutagenesis in vivo.Both prokaryotic and eukaryotic cells are endowed with multiple specialized DNA polymerases that are devoid of 3Ј35Ј proofreading exonuclease activity and replicate undamaged DNA in vitro with low fidelity and weak processivity (5). These specialized enzymes support DNA synthesis past a spectrum of template strand base damage by a process called translesion DNA synthesis (TLS), a mode of DNA damage tolerance that is fundamental to the survival of cells that suffer arrested DNA replication associated with damage to DNA.REV1 protein (which is confined to the eukaryotic kingdom) is a member of the Y family of DNA polymerases (14, 21). However, in vitro, the nucleotidyl transferase activity of REV1 is limited to the incorporation of just one or two dCMP moieties in a template-directed manner, regardless of the template nucleotide composition (19,30). This catalytic activity supports TLS past sites of base loss in vitro (19) and conceivably subserves this function in vivo. However, REV1 protein is also required for mutagenesis in both yeast and mammalian cells exposed to DNA-damaging agents that are not associated with the generation of sites of base loss, such as UV radiation (14). Remarkably, the dCMP transferase activity is dispensable for this function (1,14,18). Indeed, inactivation of the dCMP transferase activity in yeast does not result in defects in DNA damage-associated mutagenesis (9). Furthermore, a yeast mutant strain with a missense mutation in the N-terminal BRCT domain of REV1 retains dCMP transferase activity in vitro, even though it is deficient in TLS past sites of base loss and photoproducts (18).Several laboratories have demonstrated that the C-terminal ϳ100 amino acids of both mouse REV1 (mREV1) and human REV1 proteins can interact with multiple specialized DNA polymerases implicated in TLS (6,17,20,27). Additionally, different specialized DNA polymerases can compete with one another for binding to REV1 in vitro (6). Collectively, these observations suggest a presently unknown role(s) for REV1 in TLS that is unrelated to its dCMP transferase function.REV1 protein colocalizes with proliferating cell nuclear antigen (PCNA) in replication factories (27) and binds to other members of the Y family of DNA polymerases, to which it belongs, in...

REV1 protein, a eukaryotic member of the Y family of DNA polymerases, is involved in the tolerance of DNA damage by translesion DNA synthesis. It is unclear how REV1 is recruited to replication foci in cells. Here, we report that mouse REV1 can bind directly to PCNA and that monoubiquitylation of PCNA enhances this interaction. The interaction between REV1 protein and PCNA requires a functional BRCT domain located near the N terminus of the former protein. Deletion or mutational inactivation of the BRCT domain abolishes the targeting of REV1 to replication foci in unirradiated cells, but not in UV-irradiated cells. In vivo studies in both chicken DT40 cells and yeast directly support the requirement of the BRCT domain of REV1 for cell survival and DNA damage-induced mutagenesis.

Bypassing of DNA lesions by damage-tolerant DNA polymerases depends on the interaction of these enzymes with the monoubiquitylated form of the replicative clamp protein, PCNA. We have analyzed the contributions of ubiquitin and PCNA binding to damage bypass and damage-induced mutagenesis in Polymerase η (encoded by RAD30) from the budding yeast Saccharomyces cerevisiae. We report here that a ubiquitin-binding domain provides enhanced affinity for the ubiquitylated form of PCNA and is essential for in vivo function of the polymerase, but only in conjunction with a basal affinity for the unmodified clamp, mediated by a conserved PCNA interaction motif. We show that enhancement of the interaction and function in damage tolerance does not depend on the ubiquitin attachment site within PCNA. Like its mammalian homolog, budding yeast Polymerase η itself is ubiquitylated in a manner dependent on its ubiquitin-binding domain.

Pediatric glioblastoma (pGBM), although rare, is one of the leading causes of cancer-related deaths in children, with tumors essentially refractory to existing treatments. We have identified IGF1R to be a potential therapeutic target in pGBM due to gene amplification and high levels of IGF2 expression in some tumor samples, as well as constitutive receptor activation in pGBM cell lines. To evaluate the therapeutic potential of strategies targeting the receptor, we have carried out in vitro and in vivo preclinical studies using the specific IGF1R inhibitor NVP-AEW541. A modest inhibitory effect was seen in vitro, with GI 50 values of 5 to 6 mmol/L, and concurrent inhibition of receptor phosphorylation. Specific targeting of IGF1R with short interfering RNA decreased cell viability, diminished downstream signaling through phosphoinositide 3-kinase (PI3K), and induced G 1 arrest, effects mimicked by NVP-AEW541, both in the absence and presence of IGF2. Hallmarks of PI3K inhibition were observed after treatment with NVP-AEW541 by expression profiling and Western blot analysis. Phospho-receptor tyrosine kinase (RTK) arrays showed phosphorylation of platelet-derived growth factor receptor (PDGFR) a/b in pGBM cells, suggesting coactivation of an alternative RTK pathway. Treatment of KNS42 with the PDGFR inhibitor imatinib showed additional effects targeting the mitogenactivated protein kinase pathway, and cotreatment of the PDGFR inhibitor imatinib with NVP-AEW541 resulted in a highly synergistic interaction in vitro and increased efficacy after 14 days therapy in vivo compared with either agent alone. These data provide evidence that inhibition of IGF1R, in combination with other targeted agents, may be a useful and novel therapeutic strategy in pGBM.