DNA Replication, Repair, and Mutagenesis In Eukaryotic A

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，其可以通过在称为translesion DNA synthesis（TLS）的过程中穿过受损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。在大肠杆菌中，研究集中在polIV和polV及其促进TLS的能力上。在过去的一年里，我们发现pol V与细胞发生了物理相互作用？的主要重组酶，RecA，通过两个不同的机制。第一个发生时，Pol V结合RecA通过其UmuC亚基在DNA和ATP的情况下，而第二个发生通过其UmuD？在DNA和ATP的存在下，亚基，但不ATP水解。值得注意的是，pol V未能合成DNA与RecA突变体（RecA 1730），这是有缺陷的促进SOS诱变在体内，这表明RecA作为一个专性pol V辅助因子，其主要作用是激活pol V的SOS诱变。 尽管polIV和polV的主要生物学作用似乎涉及TLS，并且目前已知这两种酶都不参与碱基切除修复（BER），但我们仍然观察到脱嘌呤/脱嘧啶5？磷酸脱氧核糖（AP/5？- dRP）裂解酶活性。Pols IV和V催化磷酸二酯骨架在3？一个脱嘌呤/脱嘧啶（AP）位点的一侧，以及5？脱氧核糖磷酸（dRP）在预切开的AP位点。这两个易错聚合酶相关裂解酶的比活性约80倍小于相关的裂解酶活性的人DNA聚合酶_eta，这是一个关键的酶在短补丁BER，并表明这两个pols IV和V可能参与到迄今未确定的BER途径在大肠杆菌。 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，研究人员将酶结晶化，并通过X射线晶体学解决了聚合酶与匹配或不匹配的传入核苷酸以及错误掺入后焦磷酸产物的三元复合物的结构。这些结构表明Dpo 4可能通过其预先形成的开放活性位点拒绝错误的传入核苷酸的两种机制。首先，错配的复制碱基对导致金属离子的碱基堆积和排列不良，从而抑制掺入。其次，焦磷酸盐的缓慢释放可以通过阻止错配引物延伸和促进逆转聚合反应的焦磷酸解来增加Dpo 4的保真度。事实上，Dpo 4具有强大的焦磷酸解活性，并在焦磷酸盐存在下降解引物链。有趣的是，正确的输入核苷酸允许DNA合成克服焦磷酸解，但不正确的输入核苷酸不能。 在过去的一年中，我们确定并表征了五种新的热稳定性DPO 4样酶，以及两种重组嵌合体，与天然存在的聚合酶相比，它们具有增强的酶特性。Dpo 4样聚合酶是适度进行性的，可以在PCR中替代Taq，并且可以绕过通常阻断Taq的DNA损伤。通过使用Taq和Dpo 4酶的混合物，我们从UV照射的DNA中获得了单独使用Taq不能扩增的PCR扩增子。我们假设，在PCR反应中加入热稳定性Dpo 4样聚合酶将因此增加含损伤DNA样品的回收和分析，例如在法医或古代DNA分子应用中常见的那些。 对人类DNA聚合酶iota的研究集中于了解聚合酶在绕过紫外线诱导的光产物中的作用。对同步化野生型人S期细胞中产生的UV诱导突变谱的分析表明，只有约25%的突变发生在胸腺嘧啶（T）上，而75%的突变靶向胞嘧啶（C）。突变谱在XP-V细胞中显著变化，缺乏主要的人类TLS酶pol eta，其中约45%的突变发生在Ts处，约55%发生在Cs处。目前，还不清楚C->T突变是否实际上代表了与C相对的真正的错配，或者可能是由于在经历了脱氨基作用成为尿嘧啶（U）的UV光产物中与C相对的腺嘌呤（A）正确掺入的结果。为了评估人pol i ota在这种UV光产物的复制旁路中可能发挥的作用，我们分析了T-U环丁烷嘧啶二聚体（CPD）在体外的pol i ota依赖性旁路的效率和保真度。有趣的是，T-U CPD的多聚体依赖性旁路比相应的T-T CPD的多聚体依赖性旁路更有效地发生。鸟嘌呤（G）被错误掺入T-U CPD的3 'U对面的频率仅比正确的沃森-克里克碱基A低2倍。因此，基于我们的体外观察，我们假设pol iota通过与CPD的3 'U相对的G的频繁错误掺入而绕过T-U CPD的能力可能提供了一种机制，由此人类细胞可以降低这些病变的致突变潜力。

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 traversing 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 Escherichia coli, studies centered on polIV and polV and their ability to facilitate TLS. In the past year, we discovered that that pol V physically interacts with the cell?s main recombinase, RecA, through two distinguishable mechanisms. The first occurs when Pol V binds to RecA through its UmuC subunit in the absence of DNA and ATP, whilst the second occurs through its UmuD? subunit in the presence of DNA and ATP, but not ATP hydrolysis. Notably, pol V fails to synthesize DNA with a RecA mutant (RecA1730) that is defective in promoting SOS mutagenesis in vivo, suggesting that RecA serves as an obligate pol V accessory factor, whose principal role is to activate pol V for SOS mutagenesis. Although the their principal biological roles of polIV and polV appear to involve TLS and neither enzyme is at the present time known to be involved in base excision repair (BER), we nevertheless observed apurinic/apyrimidinc 5?-deoxyribose phosphate (AP/5?-dRP) lyase activities intrinsic to each polymerase. Pols IV and V catalyze cleavage of the phosphodiester backbone at the 3?-side of an apurinic/apyrimidinic (AP) site as well as the removal of a 5?-deoxyribose phosphate (dRP) at a preincised AP site. The specific activities of the two error-prone polymerase-associated lyases are approximately 80-fold less than the associated lyase activity of human DNA polymerase _eta, which is a key enzyme used in short patch BER and suggests that both pols IV and V may participate in a hitherto unidentified BER pathway in E.coli. 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 a matched or mismatched incoming nucleotide and with a pyrophosphate product after misincorporation. These structures suggested two mechanisms by which Dpo4 may reject a wrong incoming nucleotide with its preformed and open active site. First, a mismatched replicating base pair leads to poor base stacking and alignment of the metal ions and as a consequence, inhibits incorporation. Second, the slow release of pyrophosphate may increase the fidelity of Dpo4 by stalling mispaired primer extension and promoting pyrophosphorolysis that reverses the polymerization reaction. Indeed, Dpo4 has robust pyrophosphorolysis activity and degrades the primer strand in the presence of pyrophosphate. Interestingly, the correct incoming nucleotide allows DNA synthesis to overcome pyrophosphorolysis, but an incorrect incoming nucleotide does not. In the past year, we identified and characterized five novel thermostable Dpo4-like enzymes, as well as two recombinant chimeras that have enhanced enzymatic properties compared to the naturally occurring polymerases. The Dpo4-like polymerases are moderately processive, can substitute for Taq in PCR, and can bypass DNA lesions that normally block Taq. By using a blend of Taq and Dpo4 enzymes, we obtained a PCR amplicon from UV-irradiated DNA that was unamplifyable with Taq alone. We hypothesize that the inclusion of thermostable Dpo4-like polymerases in PCR reactions will therefore augment the recovery and analysis of lesion-containing DNA samples, such as those commonly found in forensic or ancient DNA molecular applications. Studies on human DNA polymerase iota focused on understanding the role of the polymerase in the bypass of UV-induced photoproducts. Analysis of the spectrum of UV-induced mutations generated in synchronized wild-type human S-phase cells reveals that only ~25% of mutations occur at Thymine (T), whilst 75% are targeted to Cytosine (C). The mutational spectra changes dramatically in XP-V cells, devoid of the major human TLS enzyme, pol eta, where ~45% of mutations occur at Ts and ~55% at Cs. At the present time, it is unclear whether the C->T mutations actually represent true misincorporations opposite C, or perhaps occur as the result of the correct incorporation of Adenine (A) opposite a C in a UV-photoproduct that had undergone deamination to Uracil (U). In order to assess the role that human pol iota might play in the replicative bypass of such UV-photoproducts, we analyzed the efficiency and fidelity of pol iota-dependent bypass of a T-U cyclobutane pyrimidine dimer (CPD) in vitro. Interestingly, pol iota-dependent bypass of a T-U CPD occurs more efficiently than that of a corresponding T-T CPD. Guanine (G) was misincorporated opposite the 3'U of the T-U CPD only 2-fold less frequently than the correct Watson-Crick base, A. Thus, based upon our in vitro observations, we hypothesized that the ability of pol iota to bypass T-U CPDs through the frequent misincorporation of G opposite the 3'U of the CPD, may provide a mechanism whereby human cells can decrease the mutagenic potential of these lesions.