Dna Replication, Repair, And Mutagenesis In Eukaryotic A

真核生物 A 中的 DNA 复制、修复和突变

• 批准号: 6671878
• 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/6671878
• 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 are phylogenetically related DNA polymerases that have collectively been termed the Y-family of DNA polymerases. In the past year, scientific studies within the section have focussed on understanding the molecular 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. Interestingly, these studies suggest that two distinct biochemical modes of RecA binding are necessary for pol V-catalyzed translesion replication. One RecA mode is characterized by a strong stimulation in nucleotide incorporation either directly opposite a lesion or at undamaged template sites, but by the absence of lesion bypass. A separate RecA mode is necessary for translesion synthesis Scientist within the section 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 including a propensity to make frameshift mutations. S. solfataricus Dpo4 has been overproduced, purified and its structure has 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 sufficiently large enough to accommodate significant structural rearrangements of the nascent primer terminus including primer-template misalignment and flipping of bases into the minor groove, so as to avoid the lesions in DNA. 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 on undamaged DNA in vitro, the enzyme is also highly error-prone when copying a variety of DNA lesions. An exception was at Benzo[a]pyrene diol epoxide adducts of deoxyadenosine, where the enzyme efficiently inserted the correct base, dTMP, opposite the adducted adensosine base. Further elongation was, however, limited and appears to be performed by the related Y-family polymerase, pol kappa. Based upon our in vitro observations, we hypothesize that pol iota and pol kappa act together to facilitate the error-free bypass of diol epoxide-adducted deoxyadenosine in vivo and in doing so, protect humans from the carcinogenic effects of exposure to Beno[a]pyrene diol epoxides. We have also recently examined the sub-cellular localization of pol iota within a living human cell. These studies revealed that despite the fact that pol iota lacks an obvious nuclear localization signal, it is predominantly localized to the nucleus, where it associates with the cell?s normal replication machinery. Following DNA damage, pol iota accumulates into discrete foci at sites of stalled replication forks. Interestingly, the pattern of foci formation was identical to that previously reported for the related Y-family polymerase, pol eta, suggesting that damage-induced pol iota- and pol eta-foci formation is tightly coordinated within the cell. Using the yeast two-hybrid assay, in vitro ?pull-down? assays and Far western analysis, we discovered that pol eta and pol iota interact with each other. Our data suggest, therefore, that human pols eta and iota may coexist in a larger holoenzyme complex whereby their lesion-bypassing activities can be coordinated in response to DNA damage and that both enzymes may play a general role in maintaining genomic integrity, as well as participating in translesion replication.

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是系统发育相关的DNA聚合酶，这些聚合酶统称为DNA聚合酶的Y-家庭。 在过去的一年中，本节内的科学研究的重点是理解生命三个王国中跨性别复制的分子机制：细菌，古细菌和真核细胞。在大肠杆菌中，此过程仅在UMUC与UMUD物理相互作用时才发生？形成Umud？2C，（POLV）。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。有趣的是，这些研究表明，RECA结合的两种不同的生化模式对于pol V催化的跨性别复制是必需的。一种RECA模式的特征是在核苷酸掺入直接对面或未损坏的模板位点，但由于没有病变旁路的刺激。对于跨性别的综合，需要单独的RECA模式 本节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，该蛋白质具有许多类似于其他Dinb聚合酶的生化特性，包括制造移料突变的倾向。 S. solfataricus dpo4已被过量生产，纯化，其结构已通过X射线晶体学解决了。像迄今为止表征的所有DNA聚合酶一样，酶具有类似于右手的拓扑结构，其域类似于手指？，a？palm？和一个拇指？ DPO4还拥有一个独特的域，称为“小手指”？这有助于酶与DNA结合。有趣的是，酶的活性位点足够大，足以容纳新生底漆末端的显着结构重排，包括底漆 - 板板未对准和将碱基翻转到小凹槽中，以避免DNA中的病变。 科学家最近在该节中发现的人类DNA聚合酶IOTA的研究表明，除了在体外表现出显着的模板依赖性的失误频谱外，该酶在复制各种DNA病变时也非常容易发生。在脱氧腺苷的苯并[A] pyrene二醇环氧加合物中，有一个例外，其中酶有效地插入了正确的碱DTMP，与加合的腺苷碱基相对。然而，进一步的伸长受到限制，并且似乎由相关的Y-家庭聚合酶Pol Kappa进行。基于我们的体外观察，我们假设Pol Iota和Pol Kappa共同起作用，以促进体内二醇环氧氧化物添加的脱氧腺苷的无错误旁路，并在这样做的过程中保护人类免受暴露于Beno暴露的致癌作用。 我们最近还检查了Pol Iota在活细胞中的细胞亚细胞定位。这些研究表明，尽管Pol Iota缺乏明显的核定位信号，但它主要定位于细胞核，它与细胞的正常复制机制相关联。 DNA损伤后，POL IOTA会在停滞的复制叉部位积聚成离散的焦点。有趣的是，焦点形成的模式与先前报道的相关Y-家庭聚合酶POLETA相同，这表明损伤诱导的POL IOTA和POL ETA-COCI形成在细胞内紧密地协调。使用酵母双杂交测定法，体外下拉？测定和远西方分析，我们发现Pol Eta和Pol Iota相互作用。因此，我们的数据表明，人类POLS ETA和IOTA可能会在较大的全酶复合物中共存，从而可以响应DNA损伤来协调其病变 - 型 - 型 - 型 - 并且这两种酶在维持基因组完整性方面都可能发挥一般作用，并参与转移重复。