DNA Replication, Repair, and Mutagenesis in Eukaryotic a

真核生物中的 DNA 复制、修复和突变

• 批准号: 6508761
• 负责人: ROGER WOODGATE
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6508761
• 关键词: DNA binding protein; DNA damage; DNA directed DNA polymerase; DNA repair; DNA replication; Escherichia coli; X ray crystallography; bacterial proteins; gene mutation; human tissue; molecular cloning; tissue /cell culture

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 that collectively have recently been named the Y-family of DNA polymerases. In the past year, the laboratory has focussed on mechanisms of translesion replication in all three kingdoms of life: bacteria, archaea and eukaryotic cells. In E. coli, this process only occurs when UmuC physically interacts with UmuD' to form UmuD'2C, (polV). Because polV is a low-fidelity enzyme, its activities within the cell are strictly controlled. For example, the enzyme is greatly stimulated by interactions with the RecA protein. RecA normally binds to regions of single stranded DNA and generally blocks genome duplication by replicative enzymes. However, studies revealed that polV acts as a locomotive "cowcatcher", effectively removing RecA from the single-stranded DNA while concomitantly facilitating translesion DNA synthesis. Scientist within the lab have recently identified and cloned a DinB homolog from the archaeon Sulfolobus solfataricus P2, called DNA polymerase IV (Dpo4). Characterization of the enzyme reveals that the protein possesses many biochemical properties similar to other DinB polymerases, However, in contrast to DinB polymerases which are unable to bypass a thymine-thymine cyclobutane dimer, Dpo4 bypasses the lesion efficiently. In this regard, the enzyme is more akin to the distantly related eukaryotic DNA polymerase eta (Rad30 protein). S. solfataricus Dpo4 has been overproduced, purified and its structure has recently been solved by X-ray crystallography. Like all DNA polymerases characterized to date, the enzyme possesses a topology similar to a right hand with domains that resemble "fingers", a "palm" and a "thumb". Dpo4 also possesses a unique domain called the "little finger" that helps the enzyme bind to DNA. Interestingly, the active site of the enzyme is large enough to accommodate two bases at one time, thus potentially explaining its ability to bypass thymine-thymine dimers. Studies with human DNA polymerase iota, which was recently discovered by scientist in the section, revealed that in addition to exhibiting a remarkable template-dependent misincorporation spectrum in vitro, the enzyme also possesses deoxyribose lyase activity and probably participates in a specialized form of base excision repair. A hallmark of pol iota is its ability to misinsert guanine opposite thymine at least three fold better than the "correct" base adenine. Recent studies suggest that the enzyme also exhibits a similar spectrum opposite Uracil and its derivatives. In living cells, Uracil frequently arises from the spontaneous deamination of cytosine residues. This results in an increase in spontaneous mutagenesis as the uracil base pairs with thymine, not guanine as it would if the base were cytosine, Thus, the ability of pol iota to misinsert guanosine opposite uracils (which were once cytosines), provides a potential mechanism for cells to reduce the extent of spontaneous mutagenesis caused by deamination of cytosine.

DNA中的损伤通常对基因组复制造成相当大的障碍。为了克服这种DNA复制的障碍，细胞利用专门的辅助因子，使新生DNA链的合成相对于阻塞病变。最近的研究表明，许多在translesion DNA合成的关键参与者属于一个大家族的结构相关的DNA聚合酶，发现在原核生物，古生菌和真核生物。系统发育分析表明，这些聚合酶可大致分为四类，以大肠杆菌UmuC、大肠杆菌E. coliDinB、酿酒酵母Rev 1和S.酿酒酵母Rad 30蛋白最近被统称为DNA聚合酶的Y家族。 在过去的一年里，该实验室专注于所有三个生命王国中的跨病变复制机制：细菌，古细菌和真核细胞。在大肠在大肠杆菌中，该过程仅在UmuC与UmuD'物理相互作用形成UmuD'2C（polV）时发生。由于polV是一种低保真酶，它在细胞内的活动受到严格控制。例如，该酶通过与RecA蛋白的相互作用而被极大地刺激。RecA通常与单链DNA区域结合，通常会阻止复制酶的基因组复制。然而，研究表明，polV作为一个机车“排障器”，有效地从单链DNA中去除RecA，同时促进跨病变DNA合成。 该实验室的科学家最近从古细菌Sulfolobus solfataricus P2中鉴定并克隆了一种DinB同系物，称为DNA聚合酶IV（Dpo 4）。该酶的表征表明，该蛋白质具有许多类似于其他DinB聚合酶的生物化学性质，然而，与不能绕过胸腺嘧啶-胸腺嘧啶环丁烷二聚体的DinB聚合酶相反，Dpo 4有效地绕过病变。在这方面，这种酶更类似于远亲的真核DNA聚合酶eta（Rad 30蛋白）。S. Solfataricus Dpo 4已被过量生产、纯化，并且其结构最近已通过X射线晶体学解析。与迄今为止表征的所有DNA聚合酶一样，该酶具有类似于右手的拓扑结构，其结构域类似于“手指”、“手掌”和“拇指”。Dpo 4还拥有一个独特的结构域，称为“小手指”，帮助酶与DNA结合。有趣的是，该酶的活性位点足够大，可以同时容纳两个碱基，从而可能解释其绕过胸腺嘧啶-胸腺嘧啶二聚体的能力。 本节科学家最近发现的人类DNA聚合酶iota的研究表明，除了在体外显示出显著的模板依赖性错误掺入谱外，该酶还具有脱氧核糖裂解酶活性，并可能参与一种特殊形式的碱基切除修复。Poliota的一个标志是它将鸟嘌呤错误插入胸腺嘧啶对面的能力至少比“正确的”碱基腺嘌呤好三倍。最近的研究表明，该酶也表现出类似的光谱相反尿嘧啶及其衍生物。在活细胞中，尿嘧啶经常由胞嘧啶残基的自发脱氨基作用产生。这导致自发诱变的增加，因为尿嘧啶碱基与胸腺嘧啶配对，而不是鸟嘌呤，因为如果碱基是胞嘧啶，则它将与胸腺嘧啶配对。因此，Poliota将鸟苷错误插入尿嘧啶（其曾经是胞嘧啶）对面的能力为细胞提供了降低由胞嘧啶脱氨基引起的自发诱变程度的潜在机制。