Role of cohesin in lesion bypass in DNA damaged human cells

黏连蛋白在 DNA 损伤人类细胞病变旁路中的作用

• 批准号: 9025476
• 负责人: LOUISE PRAKASH
• 金额: $ 34.33万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-16 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9025476
• 关键词: ATP phosphohydrolase; Acetylation; Acetyltransferase; Affect; Binding; Biochemical; Biological Assay; Bypass; Cell physiology; Cells; Chromatin; Chromosomal Instability; Chromosomes; Complex; DNA; DNA Damage; DNA lesion; DNA replication fork; DNA-Directed DNA Polymerase; Defect; Environmental Carcinogens; Environmental Pollutants; Eukaryotic Cell; Exposure to; Frequencies; Genes; Genome; Genome Stability; Genomic Instability; Human; In Situ; Lead; Left; Lesion; Ligation; MCM2 gene; Malignant Neoplasms; Mediating; Mitosis; Mus; Mutagenesis; Mutation; Plasmids; Play; Polymerase; Proteins; Pyrimidine Dimers; Replication Origin; Role; S Phase; Simian virus 40; Sister; Sister Chromatid; Site; Small Interfering RNA; System; Testing; Time; Transferase; UV induced; Xenopus; Zinc Fingers; base; carcinogenesis; cohesin; cohesion; dimer; egg; helicase; human DNA damage; innovation; novel; oxidative damage; prevent; public health relevance; ultraviolet damage; ultraviolet irradiation

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.

描述（由申请人提供）：模板链中的 DNA 损伤阻碍了复制叉的继续前进。人类细胞拥有许多 DNA 聚合酶 (Pol)，能够合成与此类阻塞损伤相反的 DNA，结构、生化和细胞研究表明，这些 Pols 以高度专业化的方式在跨损伤合成 (TLS) 中发挥作用。然而，有关 TLS 许多重要方面的信息（如果有的话）也很少。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留下相反 DNA 损伤的间隙中发生，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。在本提案中，我们将进行研究以检验新假设的许多方面，即粘连蛋白复合物在停滞在 DNA 损伤处的复制叉上从头组装，在该位置它介导复制叉的稳定性，并且 TLS 与停滞的复制叉。在目标 1 中，我们将在人体细胞中进行研究，以：(a) 分析黏连蛋白复合物的各种蛋白质对于 TLS 对抗紫外线诱导的 DNA 损伤的需求。对于这些研究，我们将使用基于 SV40 起源的质粒，该质粒在模板链之一上具有位点特异性损伤。诱变研究将使用人类细胞中质粒上携带的supF基因以及整合到小鼠细胞染色体中的cII基因进行； (b) 确定黏连蛋白复合物将 TLS Pols 定位到 UV 损伤细胞复制灶中的要求； (c) 确定在紫外线损伤的细胞中与复制蛋白和 TLS Pols 形成复合物所需的粘连蛋白； (d) 确定 TLS 中 Smc3 乙酰化的要求。在目标 2 中，我们将开展以下研究：(a) 检查黏连蛋白复合物在紫外线损伤的人类细胞中通过 DNA 损伤促进复制的需要； (b) 使用原位连接测定，检查在紫外线损伤的人类细胞中是否发生粘连蛋白与 TLS Pols 和 CMG 复合物的直接相互作用； (c) 通过 ChIP 分析确定 TLS 是否发生在位点特异性顺式-syn TT 二聚体上，与复制叉处的 CMG 解旋酶复合物以及粘连蛋白复合物结合； (d) 分析粘连蛋白在非洲爪蟾卵提取物系统中 TLS 中的作用。 通过协调 TLS 与在环境和细胞 DNA 损伤剂诱导的 DNA 损伤处停滞的复制叉，粘连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。根据这一观点，粘连蛋白的缺陷会导致基因组高度不稳定性，并与癌症相关。因此，粘连蛋白在人类中发挥着重要的癌症抑制作用。