Scientists within the Laboratory of Genomic Integrity (LGI) study the mechanisms by which mutations are introduced into DNA. These studies span the evolutionary spectrum and include studies in bacteria, archaea and eukaryotes. Most damage-induced mutagenesis in Escherichia coli is dependent upon the UmuD'C protein complex, which comprises DNA polymerase V (pol V). Pol V has intrinsically weak catalytic activity, but is dramatically stimulated by interactions with ATP and RecA. In a collaborative study with Myron Goodman at the University of Southern California, we have recently established three features of the roles of ATP and RecA in the activation of pol V. 1) RecA-activated DNA polymerase V (pol V Mut), is a DNA-dependent ATPase;2) bound ATP is required for DNA synthesis;3) pol V Mut function is regulated by ATP, with ATP required to bind primer-template DNA and ATP hydrolysis triggering dissociation from the DNA. Pol V Mut formed with an ATPase-deficient RecA E38K/K72R mutant hydrolyzes ATP rapidly, thereby establishing the DNA-dependent ATPase as an intrinsic property of pol V Mut distinct from the ATP hydrolytic activity of RecA when bound to single-stranded (ss)DNA as a nucleoprotein filament (RecA*). To our knowledge, no similar ATPase activity, or autoregulatory mechanism has previously been found for a DNA polymerase. Low fidelity DNA pol V 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 cells 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). In the past year, we demonstrated that RER is normally facilitated by DNA polymerase I (pol I) via classical nick translation. In vitro, pol I displaces 13 nucleotides of the RNA/DNA hybrid and through its 5'to 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 Is 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. Based upon these observations, we concluded 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. The umuC_Y11A mutant not only allows pol V to readily misincorporate ribonucleotides as frequently as deoxynucleotides, it also leaves its poor base-substitution fidelity essentially unchanged. However, the mutability of cells expressing the umuC_Y11A mutant is very low due to efficient repair mechanisms that are triggered by the misincorporated rNMPs. Comparison of the mutation frequency between strains expressing wild-type and mutant pol V therefore allowed us to identify pathways specifically directed at ribonucleotide excision repair (RER). As noted above, we have recently demonstrated that rNMPs incorporated by umuC_Y11A are efficiently removed from DNA in a repair pathway initiated by RNase HII. Indeed, in the absence of Rnase HII, umuC_Y11A-dependent mutagenesis increased from 7% of that observed with wild-type pol V to roughly 40% of wild-type levels. However, based upon the biochemical properties of umuC_Y11A, we expected to mutant to be as mutable if not more so, than wild-type pol V, suggesting the existence of additional DNA repair pathways that not only remove misincorporated ribonucleotides, but misinscorporated deoxyribonucleotides. In the past year, we discovered that mismatch repair and base excision repair only play minimal back-up roles in RER in vivo. In contrast, in the absence of functional RNase HII, umuC_Y11A-dependent mutagenesis increased significantly in uvrA, uvrB5 and uvrC strains, suggesting that rNMPs misincorporated into DNA are actively repaired by nucleotide excision repair (NER) in vivo. Participation of NER in RER was confirmed in a collaboration with Bennett van Houten at the University of Pittsburgh by reconstituting ribonucleotide-dependent NER in vitro. We showed that UvrABC nuclease-catalyzed incisions are readily made on DNA templates containing one, two, or five rNMPs and that the reactions were stimulated by the presence of mispaired bases. Similar to NER of DNA lesions, excision of rNMPs proceeds through dual incisions made at the 8th phosphodiester bond 5'and 4th-5th phosphodiester bonds 3'of the ribonucleotide. Ribonucleotides misinserted into DNA can therefore be added to the broad list of helix-distorting modifications that are substrates for NER. Our studies on the human TLS polymerases focused on the ability of a Y39A steric gate mutant of human DNA polymerase iota to incorporate ribonucleotides into DNA. This is of considerable interest, as the enzyme has been implicated in translesion DNA synthesis of both oxidative and UV-induced lesions. We discovered that human pol iota incorporates and extends NTPs opposite both damaged and undamaged template bases in a template-specific manner. The Y39A pol iota mutant was considerably more active in the presence of Mn compared with Mg and exhibited a marked increase in NTP incorporation and extension and surprisingly, it also exhibited increased dNTP base selectivity. Our results indicated that a single residue in pol iota is able to discriminate between NTPs and dNTPs during DNA synthesis. Since wild-type pol iota incorporates NTPs in a template-specific manner, we believe that certain DNA sequences may be at-risk for elevated mutagenesis during pol iota-dependent TLS. Molecular modeling indicated that the constricted active site of wild-type pol iota becomes more spacious in the Y39A variant. Therefore, the Y39A substitution not only permits incorporation of ribonucleotides, but also causes the enzyme to favor faithful Watson-Crick base pairing over mutagenic configurations.
| Vaisman, Alexandra; Woodgate, Roger (2018) Ribonucleotide discrimination by translesion synthesis DNA polymerases. Crit Rev Biochem Mol Biol 53:382-402 |
| Henrikus, Sarah S; Wood, Elizabeth A; McDonald, John P et al. (2018) DNA polymerase IV primarily operates outside of DNA replication forks in Escherichia coli. PLoS Genet 14:e1007161 |
| Vaisman, Alexandra; Woodgate, Roger (2017) Translesion DNA polymerases in eukaryotes: what makes them tick? Crit Rev Biochem Mol Biol 52:274-303 |
| Lee, Deokjae; An, Jungeun; Park, Young-Un et al. (2017) SHPRH regulates rRNA transcription by recognizing the histone code in an mTOR-dependent manner. Proc Natl Acad Sci U S A 114:E3424-E3433 |
| Frank, Ekaterina G; McLenigan, Mary P; McDonald, John P et al. (2017) DNA polymerase ?: The long and the short of it! DNA Repair (Amst) 58:47-51 |
| Frank, Ekaterina G; McDonald, John P; Yang, Wei et al. (2017) Mouse DNA polymerase ? lacking the forty-two amino acids encoded by exon-2 is catalytically inactive in vitro. DNA Repair (Amst) 50:71-76 |
| Jaszczur, Malgorzata; Bertram, Jeffrey G; Robinson, Andrew et al. (2016) Mutations for Worse or Better: Low-Fidelity DNA Synthesis by SOS DNA Polymerase V Is a Tightly Regulated Double-Edged Sword. Biochemistry 55:2309-18 |
| Maul, Robert W; MacCarthy, Thomas; Frank, Ekaterina G et al. (2016) DNA polymerase ? functions in the generation of tandem mutations during somatic hypermutation of antibody genes. J Exp Med 213:1675-83 |
| Goodman, Myron F; McDonald, John P; Jaszczur, Malgorzata M et al. (2016) Insights into the complex levels of regulation imposed on Escherichia coli DNA polymerase V. DNA Repair (Amst) 44:42-50 |
| Robinson, Andrew; McDonald, John P; Caldas, Victor E A et al. (2015) Regulation of Mutagenic DNA Polymerase V Activation in Space and Time. PLoS Genet 11:e1005482 |
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