DNA Replication, Repair, and Mutagenesis In Eukaryotic And Prokaryotic Cells

DNA复制、修复和突变（SDRRM）部分的科学家研究了将突变引入DNA的机制。这些研究传统上跨越了进化谱，包括细菌，古生菌和真核生物的研究，并涉及与世界各地的科学家合作。 作为与Andrew罗宾逊（澳大利亚卧龙岗大学）、Myron Goodman（南加州大学）和Michael考克斯（威斯康星大学麦迪逊分校）的国际科学合作的一部分，我们研究了E. coli DNA聚合酶IV（pol IV）在双链断裂修复中的作用。 为此，Andrew Robinsons小组使用带有荧光标记的pol IV的活细胞单分子显微镜，发现暴露于环丙沙星和甲氧苄啶抗生素会导致E.大肠杆菌细胞，强烈刺激pol IV活性。此外，RecA重组酶和pol IV焦点在抗生素处理后增加，并表现出强烈的共定位。有趣的是，SOS反应的诱导、RecA病灶的出现、pol IV病灶的出现和RecA-pol IV共定位都依赖于RecB功能。我们假设，pol IV病灶的定位可能反映了与RecA* 核蛋白丝的物理相互作用，该相互作用先前已在体外检测到。因此，我们的观察结果提供了一个在体内证实的pol IV在双链断裂诱导抗生素处理的细胞中的双链断裂修复的直接作用。 在与Andrew罗宾逊（澳大利亚卧龙岗大学）和Iwona Fijakowska（波兰科学院，华沙，波兰）的另一项国际科学合作中，我们在体内表征了pol VICE 391。pol VICE 391（RumA 2B）是一种低保真度聚合酶，其比相关的E. coli pol V（UmuD2C）。增强诱变的分子基础以前是未知的。使用单分子荧光显微镜观察pol V酶，我们发现诱变水平升高可能部分是由于RumB与基因组DNA的结合时间延长，导致与UmuC相比低保真度DNA合成水平增加。 已经确定pol VICE 391比pol V合成更多的DNA，我们想要研究在增加的核糖核苷酸诱导的应激条件下核糖核苷酸切除修复（RER）的分子机制。为此，我们产生了一个空间门pol VICE 391变体（pol VICE391_Y13A），它容易将核糖核苷酸错误地掺入E.大肠杆菌基因组，并使用该酶比较pol V和pol VICE 391促进的自发诱变的程度与它们各自的位阻门变体的程度。由低于野生型酶的位阻门变体促进的诱变水平指示活性RER，其从基因组中除去错误掺入的核糖核苷酸，而且还除去错误掺入的脱氧核糖核苷酸。 使用这种方法，我们证实了RNase HII在RER中起着关键作用。在不存在RNase HII的情况下，核苷酸切除修复（NER）蛋白有助于去除错误掺入的核糖核苷酸。然而，在没有RNase HII和NER的情况下发生显著的RER。大多数RNase HII和NER非依赖性RER在基因组复制期间发生在滞后链上。我们认为，这很可能是由于有效的RNase HI依赖性RER，它识别pol VICE391_Y13A产生的多核糖核苷酸束。当RNA酶HII被淹没或失活时，这些活性对于维持基因组完整性至关重要，因为表达pol VICE391_Y13A的rnhB或rnhB uvrA菌株在不存在RNA酶HII的情况下表现出基因组和质粒不稳定性。

Scientists in the Section on DNA Replication, Repair and Mutagenesis (SDRRM) study the mechanisms by which mutations are introduced into DNA. These studies have traditionally spanned the evolutionary spectrum and include studies in bacteria, archaea and eukaryotes and involve collaborations with scientists around the world. As part of an international scientific collaboration with Andrew Robinson (University of Wollongong, Australia), Myron Goodman (University of Southern California) and Michael Cox (University of Wisconsin-Madison), we investigated the role of E. coli DNA polymerase IV (pol IV) in double strand break repair. To do so, Andrew Robinsons group used live-cell single-molecule microscopy with fluorescently tagged pol IV and found that exposure to ciprofloxacin and trimethoprim antibiotics leads to the formation of double strand breaks in E. coli cells that strongly stimulate pol IV activity. Furthermore, the RecA recombinase and pol IV foci increase after antibiotic treatment and exhibit strong colocalization. Interestingly, the induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci, and RecA-pol IV colocalization, are all dependent on RecB function. We hypothesized that the positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations therefore provided an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics. In another international scientific collaboration with Andrew Robinson (University of Wollongong, Australia), and Iwona Fijakowska (Polish Academy of Sciences, Warsaw, Poland), we characterized pol VICE391 in vivo. pol VICE391 (RumA2B) is a low-fidelity polymerase that promotes considerably higher levels of spontaneous SOS-induced mutagenesis than the related E. coli pol V (UmuD2C). The molecular basis for the enhanced mutagenesis was previously unknown. Using single molecule fluorescence microscopy to visualize pol V enzymes, we discovered that the elevated levels of mutagenesis are likely due, in part, to prolonged binding of RumB to genomic DNA, leading to increased levels of low fidelity DNA synthesis compared to UmuC. Having determined that pol VICE391 synthesizes more DNA than pol V, we wanted to investigate the molecular mechanisms of Ribonucleotide Excision Repair (RER) under conditions of increased ribonucleotide-induced stress. To do so, we generated a steric gate pol VICE391 variant (pol VICE391_Y13A) that readily misincorporates ribonucleotides into the E. coli genome and have used the enzyme to compare the extent of spontaneous mutagenesis promoted by pol V and pol VICE391 to that of their respective steric gate variants. Levels of mutagenesis promoted by the steric gate variants that are lower than that of the wild-type enzyme are indicative of active RER that removes misincorporated ribonucleotides, but also misincorporated deoxyribonucleotides from the genome. Using such an approach, we confirmed that RNase HII plays a pivotal role in RER. In the absence of RNase HII, Nucleotide Excision Repair (NER) proteins help remove misincorporated ribonucleotides. However, significant RER occurs in the absence of RNase HII and NER. Most of the RNase HII and NER-independent RER occurs on the lagging strand during genome duplication. We suggested that this was most likely due to efficient RNase HI-dependent RER which recognizes the polyribonucleotide tracts generated by pol VICE391_Y13A. These activities are critical for the maintenance of genomic integrity when RNase HII is overwhelmed, or inactivated, as rnhB or rnhB uvrA strains expressing pol VICE391_Y13A exhibit genome and plasmid instability in the absence of RNase HI.