DNA Replication, Repair, and Mutagenesis In Eukaryotic

真核生物中的 DNA 复制、修复和突变

• 批准号: 6992855
• 负责人: ROGER WOODGATE
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6992855
• 关键词: Archaea; DNA damage; DNA directed DNA polymerase; DNA repair; DNA replication; X ray crystallography; active sites; bacteria; enzyme activity; eukaryote; gene mutation; human tissue; prokaryote

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.

基因组完整性实验室（LGI）中的科学家研究了将突变引入受损DNA的机制。现在众所周知，许多长期与诱变过程有关的蛋白质实际上是低保真DNA聚合酶，可以通过在称为Translesion DNA合成（TLS）的过程中移动经过受损的DNA来复制。一种这样的聚合酶POLETA中缺陷的人类患有静脉色素。它们对紫外线表现出敏感性，并且容易发生阳光引起的皮肤癌。 在过去的一年中，旨在理解Y-家庭聚合酶功能的实验跨越了进化谱，并纳入了对生命三个王国的生物体的研究。在大肠杆菌中，研究以POLV为中心及其促进转移复制的能力。 LGI中的科学家先前鉴定，克隆并表征了来自sulfolobus solfataricus P2的Dinb同源物，称为DNA聚合酶IV（DPO4）。在一项合作研究中，研究人员结晶了酶，并通过X射线晶体学求解了聚合酶的三元复合物以及无碱性位点和苯并[a] pyrene DNA加合物的结构。这些结构研究表明，酶的活性位点很大，很容易容纳通常阻断高富达复制的病变。结构研究还表明，所谓的“小手指”？ （LF）聚合酶的结构域在TLS期间经历了相当大的运动。通过制造具有不同生化特性的密切相关的DPO4和DBH聚合酶的嵌合体进一步研究了LF结构域在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结构域的起源。的确，如果它来自DPO4，则嵌合酶具有类似于DPO4的自然界，如果来自DBH，则嵌合体的表现非常类似于DBH。 对人DNA聚合酶IOTA的研究重点是了解聚合酶与增殖细胞核抗原（PCNA）之间的相互作用。 PCNA通常与细胞的高富达聚合酶相互作用，使其具有良好的加工性。 LGI中的科学家发现，PCNA还以模板依赖性的方式刺激了Pol Iota的加工性。有趣的是，POL IOTA的假定PCNA结合基序之一或PCNA的域间连接器环中的突变减少了POL IOTA和PCNA之间的结合，并同时减少PCNA依赖性刺激POL IOTA在体外的刺激。此外，在保留与体内与POL ETA相互作用的能力的同时，POL IOTA突变体在DNA损伤后未能积聚在复制灶中。由于这些发现的结果，LGI中的科学家假设PCNA既是复制中涉及的不同活动的脚手架和调节剂，似乎可以募集和协调复制和TLS-聚合酶，以确保基因组完整性。