Mechanisms of RNA-DNA repair

RNA-DNA 修复机制

基本信息

批准号：
10594960
负责人：
Richard T Pomerantz
金额：
$ 37.73万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-08 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10594960
关键词：
Animal Model B-DNA Base Excision Repairs Biochemical Biochemistry Biological Biological Assay Birds CRISPR/Cas technology Cell model Cells Cellular Assay Chimeric Proteins Collaborations Complex DNA DNA Damage DNA Primers DNA Repair DNA lesion DNA-Directed DNA Polymerase Deoxyribonucleotides Dependence Development Diameter Double Strand Break Repair Embryo Enzymes Exhibits Family Fibroblasts Genome HIV Human Immunoglobulin Class Switching Immunoglobulin Somatic Hypermutation Immunoglobulin Switch Recombination In Vitro Kinetics Knockout Mice Laboratories Length Mammalian Cell Measures Mediating Molecular Moloney Leukemia Virus Mus Nucleotides Plasmids Polymerase Publishing RNA RNA primers RNA-Directed DNA Polymerase Recombinants Reporter Reverse Transcription Ribonucleotides Role Structure Testing Virus cancer cell cancer survival deoxyribonucleoside triphosphate embryonic stem cell genome editing helicase human DNA in vitro activity in vivo novel repaired response tripolyphosphate

项目摘要

Polymerase theta (Polq) is a unique polymerase-helicase fusion protein that performs multiple functions in DNA repair in mammalian cells. Polq was initially characterized as a translesion DNA polymerase due to its ability to perform replication opposite DNA lesions in vitro, and Polq translesion synthesis (TLS) has now been confirmed in cells. Our laboratory and others have also identified an essential role for Polq in repairing double-strand breaks (DSBs) via microhomology-mediated end-joining (MMEJ). Polq has also been implicated in replication repair, base excision repair, somatic hypermutation and class-switch recombination. Whether this unique and error- prone polymerase exhibits additional activities in DNA repair remains unknown. Here, we discover that recombinant human Polq exhibits robust reverse transcriptase (RT) activity, whereas all other human DNA polymerases tested from the A, B, Y and X families fail to perform reverse transcription beyond 1-2 nucleotides. Polq generates identical reverse transcription products as retroviral RTs. Unexpectedly, Polq exhibits a significantly higher velocity and fidelity of deoxyribonucleotide incorporation on RNA versus DNA templates. These preliminary studies identify Polq as a novel RT in mammalian cells. We plan to elucidate the molecular basis of Polq RT activity in vitro and investigate multiple RNA-templated DNA repair (RNA-DNA repair) mechanisms in cells and in vivo by developing the following Aims: 1. To characterize Polq reverse transcriptase activity in vitro; 2. To elucidate the molecular basis of Polq reverse transcriptase activity; 3. To investigate RNA-DNA repair using cellular and animal models. We anticipate that these studies will confirm and characterize Polq RT activity in vitro and in a biological setting, and elucidate novel mechanisms of mammalian RNA-DNA repair.

聚合酶硫代（POLQ）是一种独特的聚合酶丝酶融合蛋白，在DNA中执行多种功能在哺乳动物细胞中修复。 POLQ最初被描述为跨质量DNA聚合酶，因为它的能力在体外执行与DNA病变相对的复制，而POLQ Translesy合成（TLS）现已确认在细胞中。我们的实验室和其他实验室还确定了POLQ在修复双链断裂中的重要作用（DSB）通过微学介导的最终连接（MMEJ）。 POLQ也与复制修复有关，基础切除修复，体外突击和类切换重组。这个独特而错误 - 俯卧的聚合酶在DNA修复中表现出其他活性仍然未知。在这里，我们发现重组人POLQ表现出强大的逆转录酶（RT）活性，而所有其他从A，B，Y和X家族测试的人DNA聚合酶无法进行反向转录超过1-2个核苷酸。 POLQ生成与逆转录病毒RT相同的逆转录产物。出乎意料的是，POLQ表现出明显更高的速度和脱氧核糖核苷酸掺入的速度和保真度 RNA与DNA模板。这些初步研究将POLQ视为哺乳动物细胞中的新RT。我们计划为了阐明体外POLQ RT活性的分子基础，并研究了多个RNA-模拟DNA修复（RNA-DNA修复）细胞和体内的机制通过开发以下目的：1。表征POLQ 体外逆转录酶活性； 2。阐明POLQ逆转录酶活性的分子基础； 3。使用细胞和动物模型研究RNA-DNA修复。我们预计这些研究将确认并在体外和生物环境中表征POLQ RT活性，并阐明哺乳动物RNA-DNA修复。