DNA polymerase (Pol) is an error-prone DNA polymerase involved in translesion DNA synthesis. Pol consists of two subunits: the catalytic REV3, which belongs to B family DNA polymerase, and the noncatalytic REV7. REV7 also interacts with REV1 polymerase, which is an error-prone Y family DNA polymerase and is also involved in translesion DNA synthesis. Cells deficient in one of the three REV proteins and those deficient in all three proteins show similar phenotype, indicating the functional collaboration of the three REV proteins. REV7 interacts with both REV3 and REV1 polymerases, but the structure of REV7 or REV3, as well as the structural and functional basis of the REV1-REV7 and REV3-REV7 interactions, remains unknown. Here we show the first crystal structure of human REV7 in complex with a fragment of human REV3 polymerase (residues 1847-1898) and reveal the mechanism underlying REV7-REV3 interaction. The structure indicates that the interaction between REV7 and REV3 creates a structural interface for REV1 binding. Furthermore, we show that the REV7-mediated interactions are responsible for DNA damage tolerance. Our results highlight the function of REV7 as an adapter protein to recruit Pol to a lesion site. REV7 is alternatively called MAD2B or MAD2L2 and also involved in various cellular functions such as signal transduction and cell cycle regulation. Our results will provide a general structural basis for understanding the REV7 interaction.Large numbers of DNA lesions occur daily in every cell, and the majority of the DNA lesions stall replicative DNA polymerases. This results in the arrest of DNA replication, which causes lethal effects including genome instability and cell death. Translesion DNA synthesis (TLS)2 releases this replication blockage by replacing the stalled replicative polymerase with a DNA polymerase specialized for TLS (TLS polymerase). It is generally considered that TLS includes two steps performed by at least two types of TLS polymerases, namely inserter and extender polymerases (reviewed in Refs. 1 and 2). In the first step, the stalled replicative polymerase is switched to an inserter polymerase such as Pol, Pol, Pol, or REV1, which are classified as Y family DNA polymerases (3) and have different lesion specificity (reviewed in Refs. 4 -8), and an inserter polymerase incorporates nucleotides opposite the DNA lesion instead of the stalled replicative polymerase. In the second step, an inserter polymerase is switched to the extender polymerase DNA polymerase (Pol), and then Pol extends a few additional nucleotides before a replicative polymerase restarts DNA replication.Pol consists of the catalytic REV3 and the noncatalytic REV7 subunits. REV3 is classified as a B family DNA polymerase on the basis of the primary sequence. The catalytic activity of yeast REV3 is stimulated by yeast REV7 (9). Biochemical analysis has been done only for yeast REV3 but not mammalian REV3, because the molecular mass of human REV3 is larger (ϳ350 kDa) than that of yeast REV3 (ϳ150 kDa). Disruption of the ...

Topoisomerase inhibitors such as camptothecin and etoposide are used as anti-cancer drugs and induce double-strand breaks (DSBs) in genomic DNA in cycling cells. These DSBs are often covalently bound with polypeptides at the 3′ and 5′ ends. Such modifications must be eliminated before DSB repair can take place, but it remains elusive which nucleases are involved in this process. Previous studies show that CtIP plays a critical role in the generation of 3′ single-strand overhang at “clean” DSBs, thus initiating homologous recombination (HR)–dependent DSB repair. To analyze the function of CtIP in detail, we conditionally disrupted the CtIP gene in the chicken DT40 cell line. We found that CtIP is essential for cellular proliferation as well as for the formation of 3′ single-strand overhang, similar to what is observed in DT40 cells deficient in the Mre11/Rad50/Nbs1 complex. We also generated DT40 cell line harboring CtIP with an alanine substitution at residue Ser332, which is required for interaction with BRCA1. Although the resulting CtIPS332A/−/− cells exhibited accumulation of RPA and Rad51 upon DNA damage, and were proficient in HR, they showed a marked hypersensitivity to camptothecin and etoposide in comparison with CtIP+/−/− cells. Finally, CtIPS332A/−/−BRCA1−/− and CtIP+/−/−BRCA1−/− showed similar sensitivities to these reagents. Taken together, our data indicate that, in addition to its function in HR, CtIP plays a role in cellular tolerance to topoisomerase inhibitors. We propose that the BRCA1-CtIP complex plays a role in the nuclease-mediated elimination of oligonucleotides covalently bound to polypeptides from DSBs, thereby facilitating subsequent DSB repair.

Clinical topoisomerase I (Top1) and II (Top2) inhibitors trap topoisomerases on DNA, thereby inducing protein-linked DNA breaks. Cancer cells resist the drugs by removing topoisomerase-DNA complexes, and repairing the drug-induced DNA double-strand breaks (DSBs) by homologous recombination (HR) and non-homologous end-joining (NHEJ). Because numerous enzymes and cofactors are involved in the removal of the topoisomerase-DNA complexes and DSB repair, it has been challenging to comprehensively analyze the relative contribution of multiple genetic pathways in vertebrate cells. Comprehending the relative contribution of individual repair factors would give insights into the lesions induced by the inhibitors and genetic determinants of response. Ultimately, this information would be useful to target specific pathways to augment the therapeutic activity of topoisomerase inhibitors. To this end, we put together 48 isogenic DT40 mutant cells deficient in DNA repair and generated one cell line deficient in autophagy (ATG5). Sensitivity profiles were established for three clinically relevant Top1 inhibitors (camptothecin and the indenoisoquinolines LMP400 and LMP776) and three topoisomerase II inhibitors (etoposide, doxorubicin and ICRF-193). Highly significant correlations were found among Top1 inhibitors as well as Top2 inhibitors, while the profiles of Top1 inhibitors were different from those of Top2 inhibitors. Most distinct repair pathways between Top1 and Top2 inhibitors include NHEJ, TDP1, TDP2, PARP1 and Fanconi Anemia genes whereas HR appears relevant especially for Top1 and to a lesser extent for Top2 inhibitors. We also found and discuss differential pathways among Top1 inhibitors and Top2 inhibitors.