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：
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
项目类别：
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

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），而其他病毒则可能减弱 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)) 是宿主细胞 DNA 复制的重要简化模型，它利用单一病毒蛋白 (LT) 进行起点识别和解旋酶功能，以及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。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，并且与这三种蛋白质的磷酸化相关，具体来说： LT 是 RPA 的第二个亚基，也是 Polprim 的第二个亚基。此处显示的初步研究表明 LT 的磷酸化（通过创建拟磷酸化突变进行评估）不会影响 LT 的大多数功能（DNA 结合、六聚化、ATP 酶、结合）。 RPA 和 Polprim，由 Polprim 刺激聚合），但它确实显着抑制 DNA 解旋酶的进展，但引物的合成缺乏独立于该 DDR 的第二条途径。 Naïve LT 和 Polprim 与从 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。该提案的两个目标是在 DDR 位点制备 RPA 和 Polprim 复合物的拟磷突变体，并评估这两种复合物的功能，就像我们对它们的功能单独以及与其他两种复合物的wt和拟磷突变结合将阐明如何以协调的方式响应DDR来抑制复制叉进展背后的详细机制。 DNA 复制应激如何成为癌症治疗和人类 PyV 感染治疗的目标。