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'2C protein complex, which comprises DNA polymerase V (pol V). Biochemical characterization of pol V has been hindered by the fact that the enzyme is notoriously difficult to purify, largely because overproduced UmuC is insoluble. In the past year, we reported a simple and efficient protocol for the rapid purification of milligram quantities of pol V from just a few liters of bacterial culture. Rather than overproducing the UmuC protein, it was instead expressed at low basal levels, while UmuD'2 was expressed in trans from a high copy-number plasmid with an inducible promoter. Based upon our success in purifying E.coli UmuC, we developed a series of plasmid vectors for the soluble expression and subsequent purification of recombinant proteins that have historically proven extremely difficult to purify from E.coli. Similar to UmuC, instead of dramatically overproducing the recombinant human protein, it was instead expressed at a low basal level that facilitates the correct folding of the recombinant protein and increases its solubility. As a consequence, highly active recombinant proteins that are traditionally difficult to purify were readily purified using standard affinity tags and conventional chromatography. We demonstrated the utility of these vectors, by expressing and purifying full-length human DNA polymerases eta, iota and nu, from E.coli and showed that the purified DNA polymerases are catalytically active in vitro. Characterization of highly purified E.coli pol V in vitro revealed that it exhibits robust activity on an SSB-coated circular DNA template in the presence of the beta clamp/gamma clamp-loading complex and a RecA nucleoprotein filament (RecA*) in trans. This strong activity was attributed to the unexpectedly high processivity of pol V Mut (a complex that formed in vitro consisting of UmuD'2CRecAATP), which was recruited to a primer terminus by Single stranded binding protein. Remarkably, under these conditions, wild-type pol V Mut efficiently incorporated ribonucleosides into DNA. A Y11A substitution in the steric gate of UmuC further reduces pol V sugar selectivity and effectively converted pol V Mut into a primer-dependent RNA polymerase that is capable of synthesizing long RNAs with a processivity comparable to that of DNA synthesis. While the Y11F substitution has a minimal effect on sugar selectivity, it resulted in an increase in spontaneous mutagenesis in vivo. In contrast, an F10L substitution increased sugar selectivity and the overall fidelity of pol V Mut. Molecular modeling analysis revealed that the branched side-chain of L10 impinges on the benzene ring of Y11, so as to constrict its movement and as a consequence, firmly closes the steric gate, which in wild-type enzyme fails to guard against rNTPs incorporation with sufficient stringency. We also analyzed the ability of three UmuC steric gate mutants (F10L, Y11A and Y11F) to facilitate translesion DNA synthesis (TLS) of a cyclobutane pyrimidine dimer (CPD) in vitro, and to promote UV-induced mutagenesis and cell survival in vivo. The pol V (UmuC_F10L) mutant discriminated against rNTP and incorrect dNTP incorporation much better than wild-type pol V and although exhibiting a reduced ability to bypass a CPD in vitro, did so with high-fidelity and consequently produced minimal UV-induced mutagenesis in vivo. In contrast, pol V (UmuC_Y11A) readily misincorporated both rNTPs and dNTPs during efficient TLS of the CPD in vitro. However, cells expressing umuD'C(Y11A) were considerably more UV-sensitive and exhibited lower levels of UV-induced mutagenesis than cells expressing wild-type umuD'C or umuD'C(Y11F). We proposed that the increased UV-sensitivity and reduced UV-mutability of umuD'C(Y11A) is due to excessive incorporation of rNTPs during TLS that are subsequently targeted for repair, rather than an inability to traverse UV-induced lesions. In a collaborative project with Philip Holliger (Medical Research Council, Cambridge, UK), we further investigated DNA polymerase substrate specificity, which is fundamental to genome integrity and to polymerase applications in biotechnology. We reported the discovery of a novel specificity checkpoint located over 25 angstroms from the active site in the polymerase thumb sub-domain. In Tgo, the replicative DNA polymerase from Thermococcus gorgonarius, we identified a single mutation (E664K) within this region that enables translesion synthesis across a template abasic site or a cyclobutane thymidine dimer. In conjunction with a classic steric gate mutation (Y409G) in the active site, E664K transforms Tgo DNA polymerase into an RNA polymerase capable of synthesizing RNAs up to 1.7 kb long. We found that E664K enables RNA synthesis by selectively increasing polymerase affinity for the non-cognate RNA:DNA duplex as well as lowering the Km for NTP incorporation. The """"""""gatekeeper"""""""" mutation therefore identifies a key, missing step in the adaptive path from DNA to RNA polymerases and defines a novel post-synthetic determinant of polymerase substrate specificity with implications for the synthesis and replication of non-cognate nucleic acid polymers.
| 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 |
| Vaisman, Alexandra; Woodgate, Roger (2015) Redundancy in ribonucleotide excision repair: Competition, compensation, and cooperation. DNA Repair (Amst) 29:74-82 |
Showing the most recent 10 out of 37 publications
