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中的病变通常会对基因组重复造成相当大的障碍。为了克服该块到DNA复制，细胞利用专门的辅助因子，可以合成与阻塞​​病变相对的新生DNA链。最近的研究表明，跨性别DNA合成的许多关键参与者属于原核生物，古细菌和真核生物中发现的大型结构相关的DNA聚合酶。对这些聚合酶的系统发育分析表明，它们可以大致细分为四组，这些组大肠杆菌，大肠杆菌大肠杆菌，酿酒酵母Rev1和酿酒酵母Rad30蛋白最近被统称为DNA聚合酶的Y-家庭。 在过去的一年中，实验室集中在生命的所有三个王国中的转化复制机制：细菌，古细菌和真核细胞。在大肠杆菌中，只有在UMUC与Umud进行物理相互作用以形成Umud'2C（POLV）时才会发生此过程。由于POLV是一种低保真酶，因此严格控制其在细胞中的活性。例如，与RECA蛋白相互作用极大地刺激了酶。 RECA通常与单链DNA的区域结合，通常通过复制酶阻断基因组重复。然而，研究表明，POLV充当机车“ cowcatcher”，有效地从单链DNA中删除了RECA，同时同时促进了跨性别的DNA合成。 实验室内的科学家最近从古sulfolobus solfataricus p2鉴定并克隆了Dinb同源物，称为DNA聚合酶IV（DPO4）。酶的表征表明，该蛋白具有许多类似于其他Dinb聚合酶的生化特性，但是与无法绕过胸腺胺 - 胸腺丁烷二聚体DPO4的DINB聚合酶相比，DPO4有效地绕过病变。在这方面，酶更类似于远距离相关的真核DNA聚合酶ETA（RAD30蛋白）。 Solefataricus DPO4已被过量生产，纯化，其结构最近通过X射线晶体学解决了。像迄今为止特征的所有DNA聚合酶一样，该酶具有类似于右手的拓扑结构，其域类似于“手指”，“棕榈”和“拇指”。 DPO4还具有一个称为“小手指”的独特域，该结构域有助于酶结合到DNA。有趣的是，酶的活性位点足够大，可以一次容纳两个碱基，从而有可能解释其绕过胸腺胸腺胺二聚体的能力。 该节科学家最近发现了对人DNA聚合酶IOTA的研究，该研究表明，除了在体外表现出显着的模板依赖性失物构建谱外，该酶还具有脱氧核糖核酸裂解酶活性，并且可能还具有专门的碱性切除修复形式。 POL IOTA的标志是它的能力能够将鸟嘌呤与胸腺氨酸相对至少三倍比“正确”的基础腺嘌呤更好。最近的研究表明，该酶也表现出相对的尿嘧啶及其衍生物的相似光谱。在活细胞中，尿嘧啶经常来自胞嘧啶残基的自发脱氨基。这会导致自发诱变的增加，因为尿嘧啶碱基与胸腺氨酸对，而不是鸟嘌呤，而不是鸟嘌呤，因此，硫氨酸是透明质酸的，因此，Pol iota能够将鸟类插入相反的尿氨酸（曾经是细胞糖苷）的能力，提供了细胞的潜在机制，可以减少自发性诱发的程度，从而降低了自发性诱发的范围。