Slowing of the polyomavirus DNA replication fork in response to DDR

DDR 导致多瘤病毒 DNA 复制叉减慢

基本信息

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
项目类别：
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

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

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项目摘要

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途径可能会衰减DDR响应（例如腺病毒），甚至可能通过DDR抑制DDR（例如EBV和Polyomavirus（Pyomavirus（Pyv））。 PYV DNA复制已成为宿主细胞DNA复制的重要简化模型，它利用单个病毒蛋白（LT）来起源识别和解旋酶功能，否则募集了细胞DNA复制蛋白来复制其病毒基因组。 PYV DNA复制所需的主要临界相互作用是LT与细胞单链DNA结合复合物（RPA）和DNA聚合酶α-蛋白酶（polprim）之间的三个相互作用，所有三个相互作用都是需要启动的所有三个相互作用。我们已经确定了活细胞和体外的条件，其中DDR可防止病毒DNA复制，并表明这是由ATR介导的，并且与这三种蛋白质中每种蛋白的磷酸化相关，特别是：LT，RPA的第二个亚基，以及Polprim的第二个亚基。初步研究表明，LT的磷酸化（通过创建磷酸化突变评估）不会影响LT的大多数功能（DNA结合，己酰胺，ATPase，ATPase，ATPase，与RPA和Polprim结合，对Polprim的刺激），但通过Polprim刺激），但是它确实在极大地抑制了DNA Helicase Progression。引物的合成在第二个途径中是明确的，而与该DDR对LT的影响无关。从DDR激活的细胞中纯化的RPA幼稚的LT和Polprim被严重抑制引物合成，这表明在复制叉时，分别通过解旋酶和延迟合成，分别通过解旋酶和启动抑制进行了协调的抑制。该提案的两个目的是在其DDR位点准备RPA和Polprim复合物的磷酸化突变，并像我们对磷酸化LT所做的那样评估这两个配合物的功能。它们的功能仅以及其他两个配合物的WT和磷光突变都将阐明如何以对DDR的协调方式抑制复制叉进展背后的详细机制。这对如何将DNA复制应激的目标置于癌症治疗和治疗人PYV感染方面的影响。