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

DNA复制，修复和突变（SDRRM）部分的科学家研究突变引入DNA的机制。这些研究传统上跨越了进化光谱，包括对细菌、古细菌和真核生物的研究，并涉及与世界各地科学家的合作。

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.