Regulation of translesion synthesis by the bacterial replisome

细菌复制体对跨损伤合成的调节

基本信息

批准号：
9064813
负责人：
Joseph J. Loparo
金额：
$ 32.63万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-05-15 至 2020-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9064813
关键词：
Address Affect Binding Biochemical Bypass Cell Death Cells Chimeric Proteins DNA DNA Damage DNA Repair DNA biosynthesis DNA lesion DNA replication fork DNA-Directed DNA Polymerase Development Escherichia coli Eukaryota Genes Genomic Instability Image In Vitro Individual Kinetics Label Laboratories Lead Left Lesion Life Measures Mediating Microscopy Modeling Molecular Conformation Mutate Mutation Nature Pathway interactions Play Polymerase Prokaryotic Cells Proteins Reaction Recruitment Activity Regulation Role SOS Response Shapes Site Son of Sevenless Proteins Speed Suggestion Techniques Time Up-Regulation Work chromosome replication disease-causing mutation gene product in vivo novel overexpression polymerization protein protein interaction public health relevance reconstitution research study response screening single molecule single-molecule FRET trafficking

项目摘要

DESCRIPTION (provided by applicant): This project utilizes novel single-molecule approaches to elucidate the mechanisms by which the bacterial replisome regulates access of translesion polymerases to the replication fork and mediates translesion synthesis (TLS) across DNA lesions that block the replisome. Proper regulation of TLS is essential because TLS polymerases are significantly more error-prone than their replicative counterparts. Improper access of TLS polymerases to the replication fork is correlated with increased mutation rates in both prokaryotes and eukaryotes. Answers to fundamental mechanistic questions regarding how TLS occurs remain obscured in ensemble biochemical experiments due to the dynamic nature of TLS polymerase exchange. This project will exploit new developments in single-molecule manipulation and imaging to detect the many structural intermediates of the replisome that transiently arise during translesion synthesis. The three specific aims are: Aim 1) Determine how the processivity factor mediates polymerase exchange. Processive DNA synthesis by the polymerases of E. coli requires interactions with the bacterial processivity factor, ß. The ß clap is believed to be a loading platform for multiple polymerases but how polymerase-clamp interactions mediate polymerase trafficking at the replication fork remains unclear. This aim will utilize single-molecule approaches to characterize the kinetics of polymerase exchange and elucidate how ß-polymerase interactions facilitate switching. In parallel, single-molecule imaging of individual fluorescently labeled polymerases will directly quantify polymerase composition and conformation on ß. Aim 2) Determine how TLS polymerases associate with the replisome and carry out TLS. TLS is believed to occur either at moving replication forks through polymerase switching reactions or in ssDNA gaps generated by the replisome translocating past the lesion and repriming synthesis downstream. In this aim, single-molecule imaging of fluorescently labeled replisome components in vitro and in cells will be used to determine how TLS polymerases interact with a replisome that has collided with a leading strand lesion. Aim 3) Identify regulators of TLS within the SOS DNA damage response. Widespread DNA damage leads to induction of the SOS DNA damage response, which results in the transcriptional upregulation of over 40 gene products involved in DNA repair and TLS. On their own, SOS levels of TLS polymerases significantly inhibit replication, leading to suggestions that TLS polymerases may slow replication to allow DNA repair to occur. Yet, strains constitutively active for the SOS response appear to grow normally, indicating that SOS gene products play a role in regulating TLS polymerase access to the fork. We will determine how high concentrations of TLS polymerases remodel the replisome and work to understand how other SOS genes further regulate TLS.

描述（由申请人提供）：该项目利用新颖的单分子方法来阐明细菌复制体调节跨损伤聚合酶进入复制叉并介导跨DNA损伤的跨损伤合成（TLS）的机制，从而阻止复制体的正确调节。 TLS 的检测非常重要，因为 TLS 聚合酶比其复制对应物更容易出错。TLS 聚合酶对复制叉的不当访问与复制叉的增加相关。由于 TLS 聚合酶交换的动态性质，有关 TLS 如何发生的基本机制问题的答案仍然不清楚，该项目将利用单分子操作和成像的新进展来检测许多突变。复制体在跨损伤合成过程中短暂出现的结构中间体是：目标 1) 确定持续性因子如何介导聚合酶的持续性 DNA 合成。大肠杆菌需要与细菌持续生长因子β相互作用。β clap被认为是多种聚合酶的装载平台，但聚合酶-钳相互作用如何介导复制叉处的聚合酶运输仍不清楚。这一目标将利用单分子方法。表征聚合酶交换的动力学并阐明 ß-聚合酶相互作用如何促进并行单分子成像。单个荧光标记的聚合酶将直接量化聚合酶的组成和构象目标 2) 确定 TLS 聚合酶如何与复制体结合并执行 TLS，相信 TLS 发生在通过聚合酶转换反应移动的复制叉中或产生的 ssDNA 间隙中。通过复制体易位经过病变并在下游重新启动合成，在这一目标中，体外和细胞内荧光标记的复制体成分的单分子成像将用于确定。 TLS 聚合酶如何与与前导链损伤碰撞的复制体相互作用目标 3) 识别 SOS DNA 损伤反应中的 TLS 调节因子广泛的 DNA 损伤会诱导 SOS DNA 损伤反应，从而导致 SOS DNA 损伤反应的转录上调。超过 40 种基因产物参与 DNA 修复和 TLS，TLS 聚合酶的 SOS 水平本身会显着抑制复制，因此有人认为 TLS 聚合酶可能会减慢复制以允许 DNA 修复发生。然而，对 SOS 反应具有组成性活性的菌株似乎生长正常，这表明 SOS 基因产物在调节 TLS 聚合酶进入叉中发挥作用。我们将确定高浓度的 TLS 聚合酶如何重塑复制体，并努力了解其他 SOS 如何进行。基因进一步调节 TLS。