Role of cohesin in lesion bypass in DNA damaged human cells

描述（由申请人提供）：模板链中的DNA损伤阻断了复制叉的持续进展。人类细胞拥有许多DNA聚合酶（pol），它们具有合成与这种阻断病变相反的DNA的能力，结构、生化和细胞研究表明，这些pol在翻译合成（TLS）中以高度特化的方式起作用。但是，关于TLS的许多重要方面的信息很少（如果有的话）。目前尚不清楚TLS是与停滞的复制叉一起发生，还是在复制后发生在相反DNA病变后留下的间隙中，以及其他重要的细胞过程是否也在促进TLS绕过病变中发挥重要作用。在本提案中，我们将开展研究来检验新假设的许多方面，即内聚蛋白复合物在DNA损伤处停滞的复制叉处重新组装，在那里它介导复制叉的稳定，并且TLS与停滞的复制叉一起发生。在Aim 1中，我们将在人类细胞中进行研究：(a)分析内聚蛋白复合物的各种蛋白质对TLS对紫外线诱导的DNA损伤的要求。对于这些研究，我们将使用基于SV40起源的质粒，该质粒在其中一条模板链上含有位点特异性病变。突变研究将在人类细胞中使用携带在质粒上的supF基因，并在小鼠细胞中将cII基因整合到染色体中；(b)确定在紫外线损伤细胞中将TLS pol定位为复制焦点所需的内聚蛋白复合物；(c)确定在紫外线损伤细胞中与复制蛋白和TLS pol形成复合物所需的内聚蛋白；(d)确定TLS中Smc3乙酰化的需求。在目标2中，我们将进行以下研究：(a)研究在紫外线损伤的人类细胞中，内聚蛋白复合体通过DNA损伤促进复制的必要性；(b)采用原位连接法，检测在紫外线损伤的人类细胞中，内聚蛋白是否与TLS pol和CMG复合物发生直接相互作用；(c)通过ChIP分析确定TLS是否与复制叉上的CMG解旋酶复合体和内聚蛋白复合体一起发生在位点特异性顺式同步TT二聚体上；(d)分析黏结蛋白在爪蟾卵提取系统中TLS中的作用。通过协调TLS与在环境和细胞DNA损伤剂诱导的DNA损伤处停滞的复制叉，内聚蛋白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。与这一观点一致的是，内聚蛋白的缺陷赋予了高度的基因组不稳定性，并与癌症有关；因此，内聚蛋白在人体中具有重要的抑癌作用。

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.