DNA Replication, Repair, and Mutagenesis In Eukaryotes

DNA中的损伤通常对基因组复制造成相当大的障碍。为了克服这种DNA复制的障碍，细胞利用专门的辅助因子，使新生DNA链的合成相对于阻塞病变。最近的研究表明，许多关键的参与者在translsion DNA合成的DNA聚合酶，已被统称为Y家族的DNA聚合酶的遗传学相关。 在过去的一年里，科学研究的重点是了解所有三个生命王国中跨病变复制的分子机制：细菌，古细菌和真核细胞。在大肠在大肠杆菌中，该过程仅在UmuC与UmuD'物理相互作用形成UmuD'2C（polV）时发生。由于polV是一种低保真度酶，其在细胞内的活性在转录水平以及多个翻译后步骤受到严格控制。事实上，最近的研究表明，在UmuD（UmuD '的致突变无活性前体）存在下，polV促进跨损伤复制的能力大大减弱。虽然UmuD'可以与自身同源二聚化以及与UmuC形成异源三聚体复合物，但它优先与UmuD异源二聚化。因此，UmuC从polV复合物中被置换，导致其聚集为无活性的不溶性沉淀物。 该部分的科学家先前从古细菌Sulfolobus solfataricus P2中鉴定并克隆了DinB同系物，称为DNA聚合酶IV（Dpo 4）。酶的表征表明，该蛋白质具有许多类似于Y家族聚合酶的真核生物成员的生化特性，包括绕过某些DNA损伤的倾向，如胸腺嘧啶-胸腺嘧啶环丁烷嘧啶二聚体。S.最近已经结晶了solfataricus Dpo 4，并且已经通过X射线晶体学解析了聚合酶与环丁烷嘧啶二聚体和引入的核苷酸的三元复合物。这些结构揭示了酶的活性位点足够大以容纳共价连接的嘧啶二聚体，并且结构研究提供了关于真核聚合酶（如pol eta）如何能够有效且准确地绕过嘧啶二聚体的模型。 用鼠DNA聚合酶iota进行的研究表明，它具有与人聚合酶相似的酶性质，因为它在体外未损伤的DNA上表现出显著的模板依赖性错误掺入谱。在尝试对小鼠Poli进行靶向破坏的过程中，该部门的科学家偶然发现，常用的129品系小鼠在Poli基因中携带单核苷酸多态性，该多态性将丝氨酸27密码子改变为琥珀终止密码子并废除聚合酶的合成。由于pol iota是一种非常容易出错的聚合酶，因此假设其参与免疫球蛋白可变基因的体细胞超突变。然而，对多聚酶缺陷型129小鼠的体细胞超突变分析显示，它们表现出正常的诱变水平和正常的突变谱。因此，似乎pol i ota不参与体细胞超突变，或者其作用是非必需的，并且已经由另一种低保真度DNA聚合酶承担。

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 at the transcriptional level as well as at multiple post-translational steps. Indeed, recent studies suggest that the ability of polV to facilitate translesion replication is greatly attenuated in the presence of UmuD (the mutagenically inactive precursor to UmuD'). Although UmuD' can homodimerize with itself as well as form a heterotrimeric complex with UmuC, it preferentially heterodimerizes with UmuD. As a consequence, UmuC is displaced from the polV complex, causing it to aggregate as an inactive insoluble precipitate. Scientist within the section previously 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 eukaryotic members of the Y-family polymerases including a propensity to bypass certain DNA lesions like a thymine-thymine cyclobutane pyrimidine dimer. S. solfataricus Dpo4 has recently been crystallized and ternary complexes of the polymerase together with a cyclobutane pyrimidine dimer and an incoming nucleotide have been solved by X-ray crystallography. These structures reveal that the active site of the enzyme is sufficiently large enough to accommodate the covalently linked pyrimidine dimer and the structural studies provide a model as to how eukaryotic polymerases, like pol eta, can efficiently and accurately bypass a pyrimidine dimer. Studies with murine DNA polymerase iota revealed that it possesses enzymatic properties similar to human pol iota in that it exhibits a remarkable template-dependent misincorporation spectrum on undamaged DNA in vitro. During attempts to make a targeted disruption of murine pol iota, scientist within the section serendipitously discovered that the commonly used 129 strain of mice carries a single nucleotide polymorphism in the Poli gene which changes the Serine 27 codon to an amber stop codon and abrogates synthesis of the polymerase. Because pol iota is an extremely error prone polymerase, it has been hypothesized to participate in somatic hypermutation of immunoglobulin variable genes. Analysis of somatic hypermutation in the pol iota-deficient 129 mice revealed, however, that they exhibit normal levels of mutagenesis and a normal mutation spectrum. Thus, it appears that either pol iota does not participate in somatic hypermutation or that its role is non-essential and has been assumed by another low-fidelity DNA polymerase.