Mechanisms of RNA-DNA repair

RNA-DNA 修复机制

• 批准号: 10336801
• 负责人: Richard T Pomerantz
• 金额: $ 36.53万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 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/10336801
• 关键词: Mechanisms; RNA; DNA; repair

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theta(Poleta，POLQ)是一种独特的聚合酶-解旋酶融合蛋白，在DNA中具有多种功能 哺乳动物细胞中的修复。POLQ最初的特征是一种跨损伤的DNA聚合酶，因为它能够 在体外进行与DNA损伤相反的复制，POLQ跨损伤合成(TLS)现已得到证实 在牢房里。我们的实验室和其他实验室也发现了POLQ在修复双链断裂中的重要作用 (DSB)通过微同源介导的末端连接(MMEJ)。POLQ也与复制修复有关， 碱基切除修复、体细胞超突变和类转换重组。无论这个独一无二的错误- 倾向聚合酶在DNA修复中表现出额外的活性，目前尚不清楚。 在这里，我们发现重组人POLQ显示出强大的逆转录酶(RT)活性， 而来自A、B、Y和X家族的所有其他人类DNA聚合酶都不能反向执行 转录超过1-2个核苷酸。POLQ产生与逆转录病毒RTS相同的逆转录产物。 出乎意料的是，POLQ显示出显著更高的脱氧核糖核苷酸掺入速度和保真度 RNA模板与DNA模板的对比。这些初步研究确定POLQ是哺乳动物细胞中的一种新的RT。我们计划 阐明POLQ逆转录酶体外活性的分子基础并研究多重RNA模板DNA修复 (RNA-DNA修复)在细胞和体内的机制，目的如下：1.表征POLQ 2.阐明POLQ逆转录酶活性的分子基础； 3.用细胞和动物模型研究RNA-DNA修复。我们预计这些研究将证实 并在体外和生物学环境中表征POLQ RT活性，并阐明新的机制 哺乳动物的RNA-DNA修复。