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: 10408848
• 负责人: THOMAS MELENDY
• 金额: $ 19.94万
• 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 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10408848
• 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），而另一些病毒可能会减弱 DDR 反应（例如腺病毒），甚至会受到 DDR 的 DNA 复制抑制（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，它利用单个病毒蛋白 (LT) 进行起源识别和解旋酶功能，并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-引物酶 (Polprim) 之间的三种相互作用，所有三种相互作用都是启动合成所必需的。我们已经在活细胞和体外确定了 DDR 阻止病毒 DNA 复制的条件，并表明这是由 ATR 介导的，并与这三种蛋白质的磷酸化相关，特别是：LT、RPA 的第二个亚基和 Polprim 的第二个亚基。本文显示的初步研究表明，LT 的磷酸化（通过产生拟磷化突变进行评估）不会影响 LT 的大多数功能（DNA 结合、六聚化、ATP 酶、与 RPA 和 Polprim 的结合、Polprim 聚合的刺激），但它确实会显着抑制 DNA 解旋酶的进展。引物的合成缺乏独立于 LT 的 DDR 效应的第二条途径。 Naïve LT 和 Polprim 以及从 DDR 激活细胞中纯化的 RPA 的引物合成受到严重抑制，这表明在复制叉处，分别通过解旋酶和引发抑制，对前导链和滞后链合成进行协调抑制。该提案的两个目的是在其 DDR 位点制备 RPA 和 Polprim 复合物的拟磷突变体，并像我们对拟磷酸 LT 所做的那样评估这两种复合物的功能。它们单独的功能以及与其他两种复合物的wt和拟磷突变的结合将阐明如何响应DDR以协调的方式抑制复制叉进展背后的详细机制。这对于如何针对癌症治疗和人类 PyV 感染的治疗产生 DNA 复制应激的影响。