Investigating the mechanisms of ribonucleotide excision repair in Escherichia coli.
Low fidelity Escherichia coli DNA polymerase V (pol V/UmuD'2C) is best characterized for its ability to perform translesion synthesis (TLS). However, in recA730 lexA(Def) strains, the enzyme is expressed under optimal conditions allowing it to compete with the cell's replicase for access to undamaged chromosomal DNA and leads to a substantial increase in spontaneous mutagenesis. We have recently shown that a Y11A substitution in the "steric gate" residue of UmuC reduces both base and sugar selectivity of pol V, but instead of generating an increased number of spontaneous mutations, strains expressing umuC_Y11A are poorly mutable in vivo. This phenotype is attributed to efficient RNase HII-initiated repair of the misincorporated ribonucleotides that concomitantly removes adjacent misincorporated deoxyribonucleotides. We have utilized the ability of the pol V steric gate mutant to promote incorporation of large numbers of errant ribonucleotides into the E. coli genome to investigate the fundamental mechanisms underlying ribonucleotide excision repair (RER). Here, we demonstrate that RER is normally facilitated by DNA polymerase I (pol I) via classical "nick translation". In vitro, pol I displaces 1-3 nucleotides of the RNA/DNA hybrid and through its 5'→3' (exo/endo) nuclease activity releases ribo- and deoxyribonucleotides from DNA. In vivo, umuC_Y11A-dependent mutagenesis changes significantly in polymerase-deficient, or proofreading-deficient polA strains, indicating a pivotal role for pol I in ribonucleotide excision repair (RER). However, there is also considerable redundancy in the RER pathway in E. coli. Pol I's strand displacement and FLAP-exo/endonuclease activities can be facilitated by alternate enzymes, while the DNA polymerization step can be assumed by high-fidelity pol III. We conclude that RNase HII and pol I normally act to minimize the genomic instability that is generated through errant ribonucleotide incorporation, but that the "nick-translation" activities encoded by the single pol I polypeptide can be undertaken by a variety of back-up enzymes.
Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA.
Alexandra
A
Vaisman
Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA.
John P
JP
McDonald
Gene Bridges GmbH, Im Neuenheimer Feld 584, 69120 Heidelberg, Germany.
Stephan
S
Noll
Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA.
Donald
D
Huston
Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA.
Gregory
G
Loeb
Department of Biological Sciences and Chemistry, University of Southern California, University Park, Los Angeles, CA 90089-2910, USA.
Myron F
MF
Goodman
Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA. Electronic address: woodgate@nih.gov.
Roger
R
Woodgate
D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism; D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism; D012265:Ribonucleotides / Q000235:genetics* / Q000378:metabolism*
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012265:Ribonucleotides; D004256:DNA Polymerase I; D004258:DNA Polymerase III; C091139:ribonuclease HII; D016914:Ribonuclease H
RNase H; Ribonucleotide excision repair; Steric gate mutant; UmuC: spontaneous mutagenesis; Y-family DNA polymerase