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 损伤常常对基因组复制造成相当大的障碍。为了克服 DNA 复制的这种阻碍，细胞利用特殊的辅助因子来合成与阻碍病变相反的新生 DNA 链。最近的研究表明，跨损伤 DNA 合成的许多关键参与者是系统发育相关的 DNA 聚合酶，这些酶统称为 DNA 聚合酶 Y 家族。 在过去的一年中，该部门的科学研究重点是了解所有三个生命王国：细菌、古细菌和真核细胞中跨损伤复制的分子机制。在大肠杆菌中，这个过程仅在 UmuC 与 UmuD 物理相互作用时发生？形成UmuD?2C，(polV)。由于 polV 是一种低保真度酶，因此其在细胞内的活性受到严格控制。例如，与 RecA 蛋白的相互作用会极大地刺激该酶。有趣的是，这些研究表明 RecA 结合的两种不同生化模式对于 pol V 催化的跨损伤复制是必需的。一种 RecA 模式的特征是在病变正对面或未损坏的模板位点强烈刺激核苷酸掺入，但不存在病变旁路。跨损伤合成需要单独的 RecA 模式 该部门的科学家最近从古细菌 Sulfolobus solfataricus P2 中鉴定并克隆了 DinB 同源物，称为 DNA 聚合酶 IV (Dpo4)。该酶的表征表明，该蛋白质具有许多与其他 DinB 聚合酶相似的生化特性，包括产生移码突变的倾向。 S. solfataricus Dpo4 已被过量生产、纯化，并通过 X 射线晶体学解析了其结构。与迄今为止鉴定的所有 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”。和一个“拇指”。 Dpo4 还拥有一个称为“小指”的独特结构域。帮助酶与 DNA 结合。有趣的是，该酶的活性位点足够大，足以容纳新生引物末端的显着结构重排，包括引物模板错位和碱基翻转到小沟中，从而避免 DNA 损伤。 该部分科学家最近发现的人类DNA聚合酶iota的研究表明，除了在体外对未损伤的DNA表现出显着的模板依赖性错误掺入谱外，该酶在复制各种DNA损伤时也非常容易出错。脱氧腺苷的苯并[a]芘二醇环氧化物加合物是一个例外，其中酶有效地插入正确的碱基 dTMP，与加合的腺苷碱基相对。然而，进一步的延伸受到限制，并且似乎是由相关的 Y 家族聚合酶 pol kappa 进行的。根据我们的体外观察，我们假设 pol iota 和 pol kappa 共同作用，促进体内二醇环氧化物加合脱氧腺苷的无差错旁路，并在此过程中保护人类免受暴露于苯并[a]芘二醇环氧化物的致癌作用。 我们最近还研究了 pol iota 在活人类细胞内的亚细胞定位。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，与细胞的正常复制机制相关。 DNA 损伤后，pol iota 在停滞的复制叉位点积聚成离散的病灶。有趣的是，病灶形成的模式与之前报道的相关 Y 家族聚合酶 pol eta 的模式相同，表明损伤诱导的 pol iota 和 pol eta 病灶形成在细胞内紧密协调。使用酵母双杂交测定，体外“下拉”？分析和远西方分析，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 可能共存于一个更大的全酶复合物中，从而可以协调它们的损伤绕过活性以响应 DNA 损伤，并且这两种酶可能在维持基因组完整性以及参与跨损伤复制方面发挥一般作用。