DNA Replication, Repair, and Mutagenesis In Eukaryotic

基因组完整性实验室（LGI）的科学家研究了突变引入受损DNA的机制。现在已知，许多长期参与诱变过程的蛋白质实际上是低保真度DNA聚合酶，它们可以通过在称为translesion DNA synthesis（TLS）的过程中移动经过受损DNA进行复制。如果一种聚合酶pol eta有缺陷，那么人类就会患上着色性干皮病;他们对紫外线表现出敏感性，并且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光谱，包括对所有三个生命王国的生物体的研究。在大肠大肠杆菌，研究集中在polV和它的能力，以促进跨病变复制。LGI的科学家们以前鉴定，克隆和表征了来自古细菌Sulfolobus solfataricus P2的DinB同系物，称为DNA聚合酶IV（Dpo 4）。在一项合作研究中，研究人员使酶结晶，并通过X射线晶体学解决了聚合酶与无碱基位点和苯并[a]芘DNA加合物的三元复合物的结构。这些结构研究表明，该酶的活性位点很大，很容易容纳通常阻断高保真复制酶的病变。结构研究还表明，所谓的？小指头？(LF)聚合酶的结构域在TLS期间经历相当大的移动。LF结构域在TLS中的关键作用通过制备表现出不同生物化学性质的密切相关的Dpo 4和Dbh聚合酶的嵌合体来进一步研究。这些研究表明，嵌合体的生化特性在很大程度上取决于LF结构域的起源。事实上，如果它来自Dpo 4，嵌合酶在性质上是Dpo 4样的，如果它来自Dbh，嵌合体的行为非常像Dbh。 对人类DNA聚合酶iota的研究集中于了解聚合酶与增殖细胞核抗原（PCNA）之间的相互作用。PCNA通常与细胞相互作用？的高保真聚合酶赋予它巨大的持续合成能力。LGI的科学家们发现，PCNA还在体外以模板依赖的方式刺激poliota的持续合成能力。有趣的是，pol iota的一个推定的PCNA结合基序或PCNA的结构域间连接环中的突变，减少了pol iota和PCNA之间的结合，并伴随着减少了体外pol iota活性的PCNA依赖性刺激。此外，虽然保留了其在体内与pol eta相互作用的能力，但pol eta突变体在DNA损伤后未能在复制灶中积累。作为这些发现的结果，LGI的科学家假设PCNA作为复制中所涉及的不同活动的支架和调节剂，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.