DNA Replication, Repair, and Mutagenesis In Eukaryotic

基因组完整性实验室(LGI)的科学家研究突变引入受损DNA的机制。现在已经知道，许多长期参与突变过程的蛋白质实际上是低保真的DNA聚合酶，可以通过在一个称为跨病变DNA合成(TLS)的过程中越过受损的DNA进行复制。其中一种聚合酶polETA缺陷的人类患有着色性干皮病；他们对紫外线敏感，容易患上阳光诱发的皮肤癌。 在过去的一年里，旨在了解Y家族聚合酶功能的实验跨越了进化的光谱，包括对所有三个生命王国的生物体的研究。在大肠杆菌中，研究集中在polV及其促进跨病变复制的能力。LGI的科学家们之前从古细菌Sulfolobus solfararicus P2中鉴定、克隆和鉴定了一个DinB同源物，称为DNA聚合酶IV(Dpo4)。在一项合作研究中，研究人员将这种酶结晶，并通过X射线结晶学解决了聚合酶与碱性位点和苯并[a]芘DNA加合物的三元络合物的结构。这些结构研究表明，该酶的活性部位很大，很容易容纳通常阻止高保真复制酶的病变。结构研究也证明了所谓的？小指？在TLS过程中，聚合酶的(Lf)结构域经历了相当大的移动。通过构建表现不同生化特性的紧密相关的DPO4和DBH聚合酶的嵌合体，进一步研究了LF域在TLS中的关键作用。这些研究表明，嵌合体的生化特性在很大程度上取决于LF域的来源。事实上，如果它来自DPO4，嵌合酶本质上是类似DPO4的，如果它来自胸径，嵌合体的行为非常像胸径。 对人类DNA聚合酶IOTA的研究主要集中在了解该聚合酶与增殖细胞核抗原之间的相互作用。增殖细胞核抗原通常与细胞相互作用？S高保真聚合酶使其具有极大的处理能力。LGI的科学家发现，在体外，增殖细胞核抗原还以一种模板依赖的方式刺激细胞的加工能力。有趣的是，Poliota的一个假定的增殖细胞核抗原结合基序或PCNA的结构域连接器环的突变，减少了Poliota与PCNA之间的结合，从而降低了体外对PoliotA活性的依赖刺激。此外，在保持其与PolETA在体内相互作用的能力的同时，Poliota突变体在DNA损伤后未能在复制区积累。作为这些发现的结果，LGI的科学家们假设，增殖细胞核抗原既是复制活动的支架，也是复制活动的调节器，似乎招募和协调复制和TLS聚合酶以确保基因组的完整性。

Scientists within the Laboratory of Genomic Integrity (LGI) study the mechanisms by which mutations are introduced into damaged DNA. It is now known that many of the proteins long implicated in the mutagenic process are, in fact, low-fidelity DNA polymerases that can replicate by moving past damaged DNA in a process termed translesion DNA synthesis (TLS). Humans with defects in one such polymerase, pol eta, are afflicted with xeroderma pigmentosum; they exhibit sensitivity to ultraviolet light and are prone to sunlight-induced skin cancers. In the past year, experiments aimed at understanding the functions of Y-family polymerases spanned the evolutionary spectrum and included studies on organisms from all three kingdoms of life. In E. coli, studies centered on polV and its ability to facilitate translesion replication. Scientists in the LGI previously identified, cloned, and characterized a DinB homolog from the archaeon Sulfolobus solfataricus P2, called DNA polymerase IV (Dpo4). In a collaborative study, researchers crystallized the enzyme and solved by X-ray crystallography the structure of ternary complexes of the polymerase together with an abasic site and a Benzo[a]pyrene DNA adduct. These structural studies revealed that the active site of the enzyme is large and readily accommodates lesions that normally block high fidelity replicases. The structural studies also demonstrated that the so-called ?little finger? (LF) domain of the polymerase undergoes considerable movement during TLS. The critical role of the LF domain in TLS was further investigated by making chimeras of the closely related Dpo4 and Dbh polymerases that exhibit different biochemical properties. These studies revealed that the biochemical properties of the chimeras were largely dependent upon the origin of the LF domain. Indeed, if it was from Dpo4, the chimeric enzyme was Dpo4-like in nature, and if it was from Dbh, the chimera behaved very much like Dbh. Studies on human DNA polymerase iota focused on understanding the interactions between the polymerase and proliferating cell nuclear antigen (PCNA). PCNA normally interacts with the cell?s high fidelity polymerase endowing it with great processivity. Scientists in the LGI discovered that PCNA also stimulates the processivity of pol iota in a template-dependent manner in vitro. Interestingly, mutations in one of the putative PCNA-binding motifs of pol iota or the interdomain connector loop of PCNA, diminish the binding between pol iota and PCNA and concomitantly reduce PCNA-dependent stimulation of pol iota activity in vitro. Furthermore, whilst retaining its capacity to interact with pol eta in vivo, the pol iota mutant failed to accumulate in replication foci after DNA damage. As a consequense of these findings, scientists in the LGI hypothesized that PCNA, acting as both a scaffold and a modulator of the different activities involved in replication, appears to recruit and coordinate replicative and TLS-polymerases to ensure genome integrity.