DNA Replication, Repair, and Mutagenesis in Eukaryotic a

DNA损伤常常对基因组复制造成相当大的阻碍。为了克服这种对DNA复制的阻碍，细胞利用专门的辅助因子，使新生DNA链的合成与阻断病变相反。最近的研究表明，翻译DNA合成的许多关键参与者属于一个结构相关的DNA聚合酶大家族，该家族存在于原核生物，古细菌和真核生物中。这些聚合酶的系统发育分析表明，它们可以大致分为四类，以大肠杆菌UmuC、大肠杆菌DinB、酿酒酵母Rev1和酿酒酵母Rad30蛋白为代表，最近被统称为y家族的DNA聚合酶。在过去的一年里，该实验室专注于研究细菌、古生菌和真核细胞这三种生物的转译复制机制。在大肠杆菌中，这一过程仅在UmuC与UmuD‘物理相互作用形成UmuD’2C （polV）时发生。因为polV是一种低保真酶，它在细胞内的活动受到严格控制。例如，这种酶通过与RecA蛋白的相互作用而受到极大的刺激。RecA通常与单链DNA区域结合，通常通过复制酶阻断基因组复制。然而，研究表明，polV作为一个机车“捕集器”，有效地从单链DNA中去除RecA，同时促进翻译DNA的合成。

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.