Dna Replication, Repair, And Mutagenesis In Eukaryotic A

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，许多跨损伤DNA合成的关键参与者是与系统发育相关的DNA聚合酶，统称为DNA聚合酶Y家族。 在过去的一年里，该部门的科学研究重点是了解细菌、古菌和真核细胞这三个生命王国中跨病变复制的分子机制。在大肠杆菌中，只有当UMUC与UmuD？发生物理作用时，这个过程才会发生。形成UmuD？2C，(PolV)。因为polV是一种低保真的酶，所以它在细胞内的活动受到严格控制。例如，该酶受到与RecA蛋白相互作用的极大刺激。有趣的是，这些研究表明，在PolV催化的跨损伤复制中，RecA结合的两种不同的生化模式是必要的。一种RecA模式的特点是强刺激核苷酸掺入，要么直接与病变相对，要么在未受损的模板位置，但没有病变旁路。跨病变合成需要单独的RecA模式 该部门的科学家最近从考古子Sulfolobus solfararicus P2中鉴定并克隆了一个DinB同源物，称为DNA聚合酶IV(Dpo4)。对该酶的鉴定表明，该蛋白具有许多类似于其他DinB聚合酶的生化性质，包括发生移码突变的倾向。对其进行了大量生产和纯化，并用X-射线晶体衍射法对其结构进行了解析。像所有迄今所描述的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？还有一个？拇指？DPO4还拥有一个独特的域名，称为？小指？这有助于酶与DNA结合。有趣的是，该酶的活性部位足够大，足以适应新出现的引物末端的重大结构重排，包括引物-模板错位和碱基翻转到小沟中，从而避免DNA中的损伤。 对科学家最近在该部分发现的人类DNA聚合酶IOTA的研究表明，除了在体外未损伤的DNA上显示出显著的模板依赖的误掺入谱外，这种酶在复制各种DNA损伤时也非常容易出错。一个例外是脱氧腺苷的苯并[a]芘二醇环氧化物加合物，该酶有效地插入正确的碱基DTMP，与加合的腺苷碱基相反。然而，进一步的延伸是有限的，似乎是由相关的Y-家族聚合酶Polkappa执行的。基于我们的体外观察，我们假设poliota和polkappa共同作用，在体内促进二醇环氧化物加成的脱氧腺苷的无错误旁路，从而保护人类免受苯并[a]芘二醇环氧化物的致癌效应。 我们最近还研究了Poliota在活的人类细胞中的亚细胞定位。这些研究表明，尽管Poliota缺乏明显的核定位信号，但它主要定位于细胞核，在那里它与细胞-S正常的复制机制相联系。DNA损伤后，Poliota在停滞的复制叉处聚集成离散的焦点。有趣的是，病灶的形成模式与之前报道的相关Y家族聚合酶polETA的模式相同，这表明损伤诱导的Poliota和PolETA焦点的形成在细胞内是紧密协调的。使用酵母双杂交试验，体外？下拉？通过分析和远距离Western分析，我们发现Poleta和Poliota是相互作用的。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体中，通过该复合体，它们的病变绕过活性可以被协调以响应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病变复制方面发挥一般作用。

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.