Slowing of the polyomavirus DNA replication fork in response to DDR

DDR 导致多瘤病毒 DNA 复制叉减慢

• 批准号: 10289169
• 负责人: THOMAS MELENDY
• 金额: $ 23.93万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-24 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10289169
• 关键词: ATP phosphohydrolase; Acetylation; Address; Adenoviruses; Affect; Antiviral Agents; Apoptosis; Area; Attenuated; BK Virus; Binding; Biochemical; Biological Assay; Cell Cycle; Cells; Cellular Stress; Complex; DNA Binding; DNA Damage; DNA Repair; DNA Replication Factor; DNA Viruses; DNA biosynthesis; DNA polymerase alpha-primase; DNA replication fork; Disease; Enzymes; Family; Genome; Goals; Human; Human Herpesvirus 4; Human Papillomavirus; Immunocompromised Host; Immunosuppression; In Vitro; Individual; Infection; JC Virus; Knowledge; Large T Antigen; Maps; Mediating; Methylation; Modeling; Modification; Mutation; Organ; Pathway interactions; Phosphorylation; Phosphorylation Site; Phosphotransferases; Polyomavirus; Polyomavirus Infections; Post-Translational Protein Processing; Process; Progressive Multifocal Leukoencephalopathy; Protein Dephosphorylation; Proteins; Recombinants; Replication Initiation; Research; Role; Series; Simplexvirus; Single-Stranded DNA; Site; Sumoylation Pathway; TP53 gene; Therapeutic; Time; Viral; Viral Genome; Viral Proteins; Virus; Virus Diseases; cancer therapy; helicase; human DNA; mutant; novel; polymerization; prevent; recruit; repaired; replication stress; response; viral DNA

Some DNA viruses utilize cellular DNA damage response (DDR) pathways to aid in viral DNA replication (e.g. HSV and HPV), while others may attenuate the DDR response (e.g. adenovirus), or even be subject to DNA replication arrest by DDR (e.g. EBV and polyomavirus (PyV)). PyV DNA replication has been an important simplified model of host cell DNA replication that utilizes a single viral protein (LT) for origin recognition and helicase function, and otherwise recruits cellular DNA replication proteins to replicate its viral genomes. The primary critical interactions required for PyV DNA replication are the three interactions between LT and the cellular single-strand DNA binding complex (RPA) and DNA polymerase alpha- primase (Polprim), all three interactions are required for synthesis to be initiated. We have identified conditions both in living cells and in vitro where DDR prevents viral DNA replication, and shown that this is mediated by ATR, and correlates with phosphorylation of each of these three proteins, specifically: LT, the second subunit of RPA, and the second subunit of Polprim. Preliminary studies shown herein demonstrate that the phosphorylation of LT (evaluated by creating phosphomimetic mutations) doesn’t affect most functions of LT (DNA binding, hexamerization, ATPase, binding to RPA and Polprim, stimulation of polymerization by Polprim), but it does dramatically inhibit DNA helicase progression. Synthesis of primers is deficient in a second pathway independent of this DDR effect on LT. Naïve LT and Polprim with RPA purified from DDR-activated cells is severely inhibited for primer synthesis, suggesting that at the replication fork there is a coordinated inhibition of both leading and lagging strand synthesis, through helicase and priming suppression, respectively. The two Aims of this proposal are to prepare phosphomimetic mutations of both the RPA and Polprim complexes at their DDR sites, and evaluate the function of these two complexes as we have done with the phosphomimetic LT. Their functions alone, as well as in conjunction with both wt and phosphomimetic mutations of both of the other complexes will elucidate the detailed mechanisms behind how replication fork progression can be inhibited in a coordinated fashion in response to DDR. This has ramifications on how DNA replication stress can be targeted for cancer treatment and for treatment of human PyV infections.

一些DNA病毒利用细胞DNA损伤反应（DDR）途径来帮助病毒DNA复制（例如HSV和HPV），而其他DNA病毒可以减弱DDR反应（例如腺病毒），或者甚至受到DDR的DNA复制阻滞（例如EBV和多瘤病毒（PyV））。PyV DNA复制是宿主细胞DNA复制的重要简化模型，其利用单个病毒蛋白（LT）进行起点识别和解旋酶功能，并且以其他方式招募细胞DNA复制蛋白来复制其病毒基因组。PyV DNA复制所需的主要关键相互作用是LT与细胞单链DNA结合复合物（RPA）和DNA聚合酶α-引发酶（Polymerase）之间的三种相互作用，所有三种相互作用都是启动合成所需的。我们已经在活细胞和体外鉴定了DDR阻止病毒DNA复制的条件，并表明这是由ATR介导的，并与这三种蛋白质中的每一种的磷酸化相关，具体地说：LT，RPA的第二亚基和Pollycoprotein的第二亚基。本文所示的初步研究表明，LT的磷酸化（通过产生磷酸化模拟突变来评估）不影响LT的大多数功能（DNA结合、六聚化、ATP酶、与RPA和Polypeptide的结合、Polypeptide对聚合的刺激），但它确实显著抑制DNA解旋酶的进展。引物的合成是缺乏在第二个途径独立的这种DDR对LT的影响。幼稚LT和Polymorphism与RPA纯化从DDR激活的细胞被严重抑制引物合成，这表明在复制叉有一个协调的抑制前导和滞后链的合成，通过解旋酶和引发抑制，分别。该建议的两个目的是在RPA和Polypeptide复合物的DDR位点制备磷酸化模拟突变，并评估这两种复合物的功能，就像我们对磷酸化模拟LT所做的那样。以及结合两种其它复合物的野生型和拟磷酸化突变，将阐明在一种复合物中如何抑制复制叉进展的详细机制。以协调一致的方式应对复员方案。这对DNA复制应激如何靶向癌症治疗和治疗人类PyV感染具有影响。