Regulation of translesion synthesis by the bacterial replisome

 描述（由申请人提供）：该项目利用新型单分子方法阐明细菌复制体调节跨损伤聚合酶进入复制叉并介导跨损伤合成（TLS）的机制，跨DNA损伤阻断复制体。TLS的适当调节是必不可少的，因为TLS聚合酶比它们的复制对应物更容易出错。TLS聚合酶对复制叉的不当访问与原核生物和真核生物中突变率的增加相关。由于TLS聚合酶交换的动态性质，关于TLS如何发生的基本机制问题的答案在合奏生化实验中仍然模糊不清。这个项目将利用单分子操作和成像的新发展来检测在translesion合成过程中瞬时出现的复制体的许多结构中间体。三个具体目标是：目标1）确定持续合成能力因子如何介导聚合酶交换。通过E.大肠杆菌需要与细菌的持续合成能力因子（proc.据信，聚合酶夹是多个聚合酶的装载平台，但聚合酶-夹相互作用如何介导聚合酶在复制叉处的运输仍不清楚。这一目标将利用单分子方法来表征聚合酶交换的动力学，并阐明β-聚合酶相互作用如何促进转换。同时，单分子成像 单个荧光标记的聚合酶将直接定量PCR上的聚合酶组成和构象。目的2）确定TLS聚合酶如何与复制体结合并进行TLS。TLS被认为发生在通过聚合酶转换反应移动复制叉处，或者发生在由复制体易位通过损伤并重新引发下游合成产生的ssDNA缺口中。在这个目标中，荧光标记的复制体成分在体外和细胞中的单分子成像将用于确定TLS聚合酶如何与复制体相互作用，该复制体与前导链病变碰撞。目的3）确定SOS DNA损伤反应中TLS的调节因子。广泛的DNA损伤导致SOS DNA损伤反应的诱导，这导致参与DNA修复和TLS的超过40种基因产物的转录上调。TLS聚合酶本身的SOS水平显著抑制复制，这表明TLS聚合酶可能会减缓复制以允许DNA修复发生。然而，组成型SOS反应活跃的菌株似乎生长正常，表明SOS基因产物在调节TLS聚合酶进入分叉中发挥作用。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基因如何进一步调节TLS。

 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.