北京基因组研究所Nuleic Acids Res解析DNA损伤

　　

　REV1是真核生物一种特殊的DNA聚合酶，其可以作为一个有效靶标用于肿瘤治疗，降低REV1水平不仅能增强肿瘤细胞对化疗的敏感性，还能有效降低肿瘤细胞耐药性的产生。前期研究显示REV1能参与跨损伤DNA合成并引发基因组突变。最近一些研究发现，在出芽酵母、鸡DT40细胞、黑腹果蝇细胞及人类细胞中REV1参与DNA双链断裂的同源重组（HR）。然而关于REV1是如何被招募到HR位点及其确切功能还很不清楚。

　　近日，中国科学院北京基因组研究所精准基因组医学重点实验室郭彩霞研究组和动物研究所生物膜国家重点实验室唐铁山研究组合作发现，在复制压力存在下，REV1和FANCD2能协同作用保护新复制的DNA链免受核酸酶的降解，该研究成果在Nucleic Acids Research在线发表。

　　该研究首先回转REV1到敲低细胞证实了REV1确实直接参与HR，进而通过特异设计的报告系统发现了REV1参与复制依赖的HR；利用激光显微辐射系统在细胞核内定点诱发DNA双链断裂（DSBs），发现REV1可以被招募到DSBs，这种招募依赖于REV1泛素结合结构域、RAD18及单泛素化的FANCD2；在RAD18敲低的稳定细胞系中表达FANCD2-Ub嵌合体蛋白会增加REV1在DSBs的招募，揭示单泛素化的FANCD2作为RAD18的下游蛋白负责招募REV1到损伤位点；此外，REV1在激光造成的DNA损伤位点处的招募还受BRCA1和BRCA2调控；特别地，利用IdU和CIdU标记新合成的DNA链，通过DNA fiber实验发现在羟基脲或喜树碱处理后，REV1能保护新生DNA链不被核酸酶降解，维持受阻复制叉的稳定。

　　该研究成果表明REV1在DNA损伤应答中发挥多种重要功能，完善了其作为肿瘤治疗靶标的理论基础。

　　该工作受到中国科学院、科技部和国家自然科学基金委的资助，得到了北京蛋白质组研究中心研究员裴华东、西南医学中心放射肿瘤系Benjamin Chen和荷兰莱顿大学Niles de Wind课题组等的大力支持。

REV1保护新生DNA链免受核酸酶降解

原文摘要：

FANCD2 and REV1 cooperate in the protection of nascent DNA strands in response to replication stress

REV1 is a eukaryotic member of the Y-family of DNA polymerases involved in translesion DNA synthesis and genome mutagenesis. Recently, REV1 is also found to function in homologous recombination. However, it remains unclear how REV1 is recruited to the sites where homologous recombination is processed. Here, we report that loss of mammalian REV1 results in a specific defect in replication-associated gene conversion. We found that REV1 is targeted to laser-induced DNA damage stripes in a manner dependent on its ubiquitin-binding motifs, on RAD18, and on monoubiquitinated FANCD2 (FANCD2-mUb) that associates with REV1. Expression of a FANCD2-Ub chimeric protein in RAD18-depleted cells enhances REV1 assembly at laser-damaged sites, suggesting that FANCD2-mUb functions downstream of RAD18 to recruit REV1 to DNA breaks. Consistent with this suggestion we found that REV1 and FANCD2 are epistatic with respect to sensitivity to the double-strand break-inducer camptothecin. REV1 enrichment at DNA damage stripes also partially depends on BRCA1 and BRCA2, components of the FANCD2/BRCA supercomplex. Intriguingly, analogous to FANCD2-mUb and BRCA1/BRCA2, REV1 plays an unexpected role in protecting nascent replication tracts from degradation by stabilizing RAD51 filaments. Collectively these data suggest that REV1 plays multiple roles at stalled replication forks in response to replication stress.