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: 8693330
• 负责人: LOUISE PRAKASH
• 金额: $ 35.47万
• 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/8693330
• 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-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; ATP phosphohydrolase; Acetylation; Acetyltransferase; Affect; Binding; Biochemical; Biological Assay; Bypass; Cell physiology; Cells; Chromatin; Chromosomal Instability; Chromosomes; Complex; DNA; DNA Damage; DNA lesion; 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病变阻断复制叉的持续进展。人类细胞具有许多DNA聚合酶（POLS），具有与此类阻断病变相对的DNA的能力，结构，生化和细胞研究表明，这些POL以高度专业化的方式在Translesion合成（TLS）中起作用。但是，关于TLS的许多重要方面的信息几乎没有（如果有的话）。目前尚不清楚TLS是否与停滞的复制叉结合发生，还是在遗留的间隙中发生的，后者遗留在相反的DNA病变中，以及其他重要的细胞过程是否在促进TLS促进病变旁路中也起着至关重要的作用。在这项提案中，我们将进行研究，以研究新假设的许多方面，即粘蛋白复合物在复制叉处从从头开始，在DNA病变处停滞了，在DNA病变中它介导了复制叉的稳定性，而TLS则与停滞的重复叉相结合。在AIM 1中，W将在人类细胞中进行研究：（a）分析粘连蛋白复合物的各种蛋白质对紫外线诱导的DNA病变的需求。对于这些研究，我们将使用基于SV40的基于SV40的质粒，该质粒在其中一个模板链上携带了特定于位点的病变。将使用在人类细胞中的质粒上携带的SUPF基因，以及将CII基因整合到小鼠细胞中的染色体中。 （b）确定粘蛋白复合物对将TLS POL定位到紫外线受损细胞中复制灶中的需求； （c）确定粘着蛋白与复制蛋白的复杂形成以及紫外线受损细胞中的TLS POL的需求； （d）确定TLS中SMC3乙酰化的需求。在AIM 2中，我们将进行研究：（a）检查粘蛋白复合物通过紫外线受损的人类细胞中DNA病变促进复制的需求； （b）使用原位连接测定，检查粘蛋白蛋白与TLS POL的直接相互作用以及与CMG复合物的直接相互作用是否发生在紫外线受损的人类细胞中； （c）通过芯片分析确定是否在位点特异性的顺式synn TT二聚体与复制叉和粘蛋白复合物的CMG解旋酶复合物结合使用； （d）分析粘着蛋白在TLS中的作用。 通过与环境和细胞DNA损伤剂诱导的DNA病变停滞的复制叉通过协调TLS，粘着蛋白复合物将在维持基因组的完整性和忠诚度中起重要作用。为了与这个想法保持一致，粘着蛋白蛋白的缺陷赋予了高度的基因组不稳定性，并且与癌症有关。因此，粘着蛋白在人类中起重要的癌症抑制作用。