Regulation of translesion synthesis by the bacterial replisome

 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病变聚合酶对复制叉的访问并介导跨病变合成(TLS)的机制，从而阻止复制体。对TLS的适当调控是至关重要的，因为TLS聚合酶明显比它们的复制对应物更容易出错。TLS聚合酶对复制分叉的不正确访问与原核生物和真核生物中突变率的增加有关。由于TLS聚合酶交换的动态性质，在整体生化实验中，关于TLS如何发生的基本机制问题的答案仍然模糊不清。该项目将利用单分子操作和成像方面的新进展，以检测在跨损伤合成过程中瞬时出现的复制体的许多结构中间体。这三个具体目标是：目的1)确定加工性因子如何介导聚合酶交换。大肠杆菌聚合酶的过程性DNA合成需要与细菌的过程性因子进行相互作用。Bclap被认为是多个聚合酶的装载平台，但聚合酶-钳子相互作用如何在复制叉处介导聚合酶运输仍不清楚。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ç-聚合酶相互作用如何促进转换。并行的单分子成像 单个荧光标记聚合酶的组成和构象将直接定量。目的2)确定TLS聚合酶如何与复制体结合并进行TLS。TLS被认为要么发生在通过聚合酶交换反应移动的复制叉处，要么发生在复制体移位经过病变并重新启动下游合成所产生的单链DNA缺口中。在这个目标中，将使用在体外和细胞中荧光标记的复制体成分的单分子成像来确定TLS聚合酶如何与与领先链损伤相碰撞的复制体相互作用。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，导致参与DNA修复和TLS的40多个基因产物转录上调。就其本身而言，TLS聚合酶的SOS水平显著抑制复制，从而导致TLS聚合酶可能减缓复制以允许DNA修复发生。然而，具有SOS反应活性的菌株似乎生长正常，这表明SOS基因产物在调节TLS聚合酶进入叉子的过程中发挥了作用。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.