DNA Replication, Repair, and Mutagenesis In Eukaryotic A

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，可以通过穿越受损的DNA进行复制，这个过程被称为跨病变DNA合成(TLS)。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。 我们在大肠杆菌中的研究集中在PolV？S促进TLS的能力上，并作为与南加州大学Myron Goodman合作研究的一部分进行。特别是，我们研究了RecA和polV之间相互作用的性质。以前人们认为RecA与polV复制的受损DNA模板链结合。然而，值得注意的是，我们最近的研究表明，在有或没有β-加工力钳的情况下，polV催化的跨损伤合成只有当RecA核蛋白细丝组装在单独的单链(Ss)DNA分子上时才会发生。反式DNA上3‘-近端的RecA细丝末端对于刺激是必不可少的，并被沿着反式核蛋白细丝发生在其他地方的进一步的PolV-RecA相互作用所加强。基于这些观察，我们认为RecA与ssDNA结合对polV的反式刺激反映了一种独特的突变调节机制，该机制解决了RecA细丝组装在受损的模板链上阻碍跨损伤DNA合成的悖论，尽管RecA对SOS突变是绝对需要的。 作为我们在Archaea的研究的一部分，我们鉴定并鉴定了五种新的耐热DPO4样酶，以及两种重组嵌合体，与自然存在的聚合酶相比，它们的酶性质有所增强。类DPO4聚合酶是中度进行性的，可以替代聚合酶链式反应中的Taq，并且可以绕过通常阻断Taq的DNA损伤。通过使用Taq和Dpo4酶的混合物，我们从紫外线照射的DNA中获得了一种单独使用Taq不能扩增的PCR扩增产物。我们假设，在PCR反应中包括耐热的DPO4样聚合酶将因此增强对含有病变的DNA样本的恢复和分析，例如那些在法医或古代DNA分子应用中常见的DNA样本。 关于人类DNA聚合酶ETA和IOTA的研究主要集中在细胞如何调节低保真度的聚合酶，以尽量减少它们无意中接触到引物末端。作为这些研究的一部分，我们发现人类细胞使用的一种机制依赖于DNA聚合酶IOTA和ETA与泛素(Ub)之间的特定和直接的相互作用。事实上，我们发现这两种聚合酶都与游离的PolyUb链以及单一泛素化的增殖细胞核抗原(Ub-PCNA)非共价相互作用。Poliota(P692R)和polETA(H654A)的突变体被分离出来，它们与PolyUb和Ub-PCNA的相互作用存在缺陷，但保留了与未修饰的PCNA相互作用的能力。有趣的是，聚合酶突变体对DNA损伤的反应显示出显著较低的复制焦点水平，从而突显了聚合酶-Ub相互作用在调节TLS聚合酶对体内停滞的复制叉子的访问方面的生物学重要性。 在体外对人类DNA聚合酶IOTA的研究集中在了解它在紫外线诱导的光产物旁路中的作用。对同步野生型人S期细胞紫外线诱导突变的光谱分析表明，只有25%的突变发生在胸腺嘧啶(T)，而75%的突变发生在胞嘧啶(C)。在XP-V细胞中，突变谱发生了巨大的变化，缺乏人类主要的TLS酶polETA，其中约45%的突变发生在Ts，约55%的突变发生在Cs。目前，尚不清楚C-&gt；T突变实际上是否代表与C相反的真正错误结合，或者可能是由于在经过脱氨为尿嘧啶(U)的紫外光产品中正确地结合了与C相反的腺嘌呤(A)而发生的。为了评估人端粒酶在这类紫外光产物复制旁路中的作用，我们在体外分析了端粒酶依赖的T-U环丁烷嘧啶二聚体(CPD)的效率和保真度。有趣的是，与相应的T-T CPD相比，T-U CPD的转流更有效。鸟嘌呤(G)与T-U CPD的3‘U相对的错配频率仅比正确的Watson-Crick碱基A低2倍。因此，根据我们的体外观察，我们假设Poliota通过频繁的G与CPD的3’U的错配绕过T-U CPD的能力，可能提供了一种人类细胞降低这些病变的突变潜力的机制。

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. Our studies in Escherichia coli centered on polV?s ability to facilitate TLS and were performed as part of a collaborative study with Myron Goodman at the University of Southern California. In particular, we investigated the nature of the interactions between RecA and polV. It had previously been assumed that RecA binds to the damaged DNA template strand being copied by polV. Remarkably, however, our recent studies revealed that polV-catalyzed translesion synthesis, in the presence or absence of the beta-processivity-clamp, occurs only when RecA nucleoprotein filaments assemble on separate single-stranded (ss)DNA molecules in trans. A 3'-proximal RecA filament end on trans DNA is essential for stimulation and is strengthened by further polV-RecA interactions occurring elsewhere along a trans nucleoprotein filament. Based upon these observations, we suggested that trans-stimulation of polV by RecA bound to ssDNA reflects a distinctive regulatory mechanism of mutation that resolves the paradox of RecA filaments assembled in cis on a damaged template strand obstructing translesion DNA synthesis, despite the absolute requirement of RecA for SOS mutagenesis. As part of our studies in Archaea, 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 human DNA polymerases eta and iota focussed on how cells regulate the low-fidelity polymerases so as to minimize their inadvertent access to primer-termini. As part of these studies, we discoverd that one mechanism employed by human cells relies on a specific and direct interaction between DNA polymerases iota and eta with ubiquitin (Ub). Indeed, we showed that both polymerases interact noncovalently with free polyUb chains, as well as mono-ubiquitinated proliferating cell nuclear antigen (Ub-PCNA). Mutants of pol iota (P692R) and pol eta (H654A) were isolated that were defective in their interactions with polyUb and Ub-PCNA, whilst retaining their ability to interact with unmodified PCNA. Interestingly, the polymerase mutants exhibited significantly lower levels of replication foci in response to DNA damage, thereby highlighting the biological importance of the polymerase-Ub interaction in regulating the access of the TLS polymerases to stalled replication forks in vivo. In vitro studies with human DNA polymerase iota focused on understanding its role 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 occurred 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.