Role of RNase H enzymes in maintaining genome stability in Escherichia coli expressing a steric-gate mutant of pol V ICE391

• Published in: DNA repair Vol. 84; p. 102685
• Main Authors: Walsh, Erin, Henrikus, Sarah S, Vaisman, Alexandra, Makiela-Dzbenska, Karolina, Armstrong, Thomas J, Łazowski, Krystian, McDonald, John P, Goodman, Myron F, van Oijen, Antoine M, Jonczyk, Piotr, Fijalkowska, Iwona J, Robinson, Andrew, Woodgate, Roger
• Format: Journal Article
• Language: English
• Published: Netherlands 01.12.2019
• Subjects: DNA Repair; DNA-Directed DNA Polymerase - chemistry; DNA-Directed DNA Polymerase - genetics; DNA-Directed DNA Polymerase - metabolism; Escherichia coli; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Genomic Instability; Mutation; Protein Domains; Ribonucleotides - genetics; Ribonucleotides - metabolism; Translation; Ribonucleotide excision repair; DNA polymerase V; Y-family DNA polymerase
• ISSN: 1568-7856

pol V (RumA' B) is a low-fidelity polymerase that promotes considerably higher levels of spontaneous "SOS-induced" mutagenesis than the related E. coli pol V (UmuD' C). 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 DNA synthesis compared to UmuC. We have generated a steric gate pol V variant (pol V _Y13A) that readily misincorporates ribonucleotides into the E. coli genome and have used the enzyme to investigate the molecular mechanisms of Ribonucleotide Excision Repair (RER) under conditions of increased ribonucleotide-induced stress. To do so, we compared the extent of spontaneous mutagenesis promoted by pol V and pol V 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 suggest that this is most likely due to efficient RNase HI-dependent RER which recognizes the polyribonucleotide tracts generated by pol V _Y13A. These activities are critical for the maintenance of genomic integrity when RNase HII is overwhelmed, or inactivated, as ΔrnhB or ΔrnhB ΔuvrA strains expressing pol V _Y13A exhibit genome and plasmid instability in the absence of RNase HI.