DNA REPLICATION, REPAIR, AND MUTAGENESIS IN EUKARYOTIC AND PROKARYOTIC CELLS

DNA中的损伤经常对基因组复制造成相当大的障碍。为了克服这种对DNA复制的阻碍，细胞利用专门的辅助因子，允许合成与阻断损伤相对的新生DNA链。最近的研究表明，跨损伤DNA合成的许多关键参与者属于结构相关的DNA聚合酶大家族，这些聚合酶存在于原核生物、古生物和真核生物中。对这些聚合酶的系统发育分析表明，它们大致可分为四个亚类，分别为Escherichia coliUMUC、E.coliDinB、Saccharmycescerevisiae Rev1和S.cerevisiae Rad30蛋白。在过去的一年里，实验室专注于进化光谱两端的跨病变复制机制。在大肠杆菌中，只有当UMUC与UmuD‘物理相互作用形成UmuD’2C(PolV)时，才会发生这一过程。因为PolV是S的一种低保真酶，它在细胞内的活动受到严格控制。实验室的研究表明，polV的UmuD‘亚单位是ClpXP丝氨酸蛋白酶通过一种新的“反式靶向”机制专门降解的：通过这一过程，降解信号实际上位于一种实际上并未被酶降解的蛋白质中。由于细胞含有非常低水平的polV，因此必须有一种机制，使这些分子定位于染色体上它们可以进行跨病变复制的区域。基因分析表明，这种靶向是通过与RecA的相互作用实现的。事实上，这种靶向可以通过电子显微镜观察到，电子显微镜显示，当存在低浓度时，polV特异性地结合到RecA核蛋白细丝的末端。用polV进行的生化研究也揭示了对E.coliPolIII全酶以及E.coli单链结合蛋白的β和伽马复合体的需求。正在进行的研究旨在了解所有polV、RecA、β/Gamma复合体和SSB蛋白相互作用以促进跨病变复制的确切机制。对人酶的研究表明，大多数跨病变复制是由DNA聚合酶ETA执行的。然而，该实验室发现了一种相关的聚合酶，称为Poliota。有趣的是，与PolETA高效准确复制胸腺嘧啶-胸腺嘧啶环丁烷嘧啶二聚体不同，PolIOTA绕过嘧啶二聚体的能力相当有限，但非常容易出错。Poliota的生化特征表明，在未损坏的DNA模板上也容易出错。平均而言，Poliota每复制100个核苷酸就会出现一个错误。然而，酶的保真度也显著地依赖于被复制的模板碱基。例如，鸟苷与胸腺嘧啶相对插入的效果至少是“正确的”碱基腺苷的三倍。正在进行的研究旨在阐明这种显着的聚合酶在细胞内的功能。

Lesions in DNA often pose considerable impediments to genome duplication. To overcome this block to DNA replication, cells utilize specialized accessory factors that allow synthesis of nascent DNA chains opposite the blocking lesion. Recent studies suggest that many of the key participants in translesion DNA synthesis belong to a large family of structurally related DNA polymerases that are found in prokaryotes, archaea and eukaryotes. Phylogenetic analysis of these polymerases suggest that they can be broadly subdivided into four groups typified by Escherichia coli UmuC, E. coli DinB, Saccharomyces cerevisiae Rev1 and the S. cerevisiae Rad30 protein. In the past year, the laboratory has focussed on mechanisms of translesion replication at both ends of the evolutionary spectrum. In E. coli, this process only occurs when UmuC physically interacts with UmuD' to form UmuD'2C, (polV). Because polV is s a low-fidelity enzyme, its activities within the cell are strictly controlled. Studies from the lab revealed that the UmuD' subunit of polV is specifically targeted for degradation by the ClpXP serine protease by a novel mechanism of "trans-targeting": a process whereby the signal for degradation is in fact located in a protein that is not actually degraded by the enzyme. Since cells contain very low levels of polV, there must be a mechanism whereby these molecules are targeted to regions of the chromosome where they can perform translesion replication. Genetic analysis suggested that such targeting is achieved via an interaction with RecA. Indeed, such targeting was visualized by electron microscopy which revealed that when present at low concentrations, polV specifically binds to the tip of a RecA nucleoprotein filament. Biochemical studies with polV also reveal a requirement for the beta and gamma complexes from E. coli pol III holoenzyme as well as the E. coli single stranded-binding protein. Ongoing studies aim to understand the precise mechanism by which all the polV, RecA, beta/gamma complex and Ssb proteins interact to facilitate translesion replication. Studies with human enzymes suggest that most translesion replication is performed by DNA polymerase eta. The lab has, however, discovered a related polymerase, called pol iota. Interestingly, unlike pol eta which replicates thymine-thymine cyclobutane pyrimidine dimers efficiently and accurately, pol iota's ability to bypass pyrimidine dimers is quite limited, but is highly error-prone. Biochemical characterization of pol iota reveals that is also "error-prone" on undamaged DNA templates. On average, pol iota makes one error every 100 nucleotides replicated. However, the fidelity of the enzyme is also strikingly dependent upon the template base being replicated. For example, guanosine is inserted opposite thymine at least three fold better than the "correct" base adenosine. Ongoing studies are aimed at elucidating the function of this remarkable polymerase within the cell.