Role of cohesin in lesion bypass in DNA damaged human cells

描述（由申请方提供）：模板链中的DNA损伤阻断复制叉的继续进展。人类细胞具有许多DNA聚合酶（Pos），其具有合成与这种阻断病变相对的DNA的能力，并且结构、生物化学和细胞研究已经表明这些Pos以高度特化的方式在跨病变合成（TLS）中起作用。然而，关于TLS的许多重要方面的信息很少。目前尚不清楚TLS是否与停滞的复制叉一起发生，或者在复制后发生在相对DNA损伤后面留下的间隙中，以及其他重要的细胞过程是否也在促进TLS绕过损伤中发挥重要作用。在这项提案中，我们将进行研究，以检查新的假设，即粘附素复合物重新组装在复制叉停滞在DNA损伤，它介导的稳定的复制叉，TLS发生在与停滞的复制叉的许多方面。在目标1中，我们将在人类细胞中进行研究，以：（a）分析TLS对UV诱导的DNA损伤的粘附素复合物的各种蛋白质的需求。对于这些研究，我们将使用基于SV 40来源的质粒，该质粒在其中一条模板链上具有位点特异性损伤。使用人细胞中质粒上携带的supF基因和小鼠细胞中整合到染色体中的cII基因进行诱变研究;（B）确定在UV损伤细胞中将TLS Pos定位到复制灶中所需的粘着蛋白复合物;（c）确定在UV损伤细胞中与复制蛋白和TLS Pos形成复合物所需的粘着蛋白;以及（d）确定TLS中Smc 3乙酰化的要求。在目的2中，我们将进行以下研究：（a）检查在UV损伤的人细胞中通过DNA损伤促进复制对粘着蛋白复合物的需要;（B）使用原位连接测定，检查在UV损伤的人细胞中粘着蛋白与TLS Pos和与CMG复合物的直接相互作用是否发生;（c）通过ChIP分析确定TLS是否发生在与复制叉处的CMG解旋酶复合物和粘着蛋白复合物结合的位点特异性顺式-syn TT二聚体处;和（d）分析粘着蛋白在非洲爪蟾卵提取物系统中TLS中的作用。 通过协调TLS与停滞在由环境和细胞DNA损伤剂诱导的DNA损伤处的复制叉，粘附素复合物将在维持基因组的完整性和保真度方面发挥重要作用。与这一想法一致，粘附蛋白的缺陷赋予高度的基因组不稳定性并与癌症相关;因此，粘附蛋白在人类中发挥重要的癌症抑制作用。

DESCRIPTION (provided by applicant): DNA lesions in the template strand block the continued progression of the replication fork. Human cells possess a number of DNA polymerases (Pols) with the ability to synthesize DNA opposite such blocking lesions, and structural, biochemical, and cellular studies have indicated that these Pols function in translesion synthesis (TLS) in a highly specialized manner. However, there exists little, if any, information on many of the important aspects of TLS. It remains unclear whether TLS occurs in conjunction with the stalled replication fork or occurs post-replicatively in gaps that are left behind opposite DNA lesions, and whether other important cellular processes also play an essential role in promoting lesion bypass by TLS. In this proposal we will carry out studies to examine the many aspects of the novel hypothesis that the cohesin complex assembles de novo at the replication fork stalled at a DNA lesion where it mediates the stabilization of the replication fork, and that TLS occurs in conjunction with the stalled replication fork. In Aim 1, w will carry out studies in human cells to: (a) analyze the requirements of various proteins of the cohesin complex for TLS opposite UV induced DNA lesions. For these studies, we will use the SV40 origin-based based plasmid which harbors a site-specific lesion on one of the template strands. Mutagenesis studies will be done using the supF gene carried on a plasmid in human cells, and with the cII gene integrated into the chromosome in mouse cells; (b) determine the requirement of the cohesin complex for the localization of TLS Pols into replication foci in UV damaged cells; (c) determine the requirement of cohesin for complex formation with replication proteins and with TLS Pols in UV damaged cells; and (d) determine the requirement of Smc3 acetylation in TLS. In Aim 2, we will carry out studies to: (a) examine the requirement of the cohesin complex for promoting replication through DNA lesions in UV damaged human cells; (b) using in situ ligation assays, examine whether direct interactions of cohesin proteins with TLS Pols and with the CMG complex occur in UV damaged human cells; (c) determine by ChIP analyses whether TLS occurs at a site-specific cis-syn TT dimer in conjunction with the CMG helicase complex at the replication fork and with the cohesin complex; and (d) analyze the role of cohesin in TLS in the Xenopus egg extract system. By coordinating TLS with the replication fork stalled at DNA lesions induced by environmental and cellular DNA damaging agents, the cohesin complex would play an important role in maintaining the integrity and fidelity of the genome. In keeping with this idea, defects in the cohesin proteins confer a high degree of genomic instability and are associated with cancers; thus, the cohesin proteins play an important cancer suppressor role in humans.