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 globe. As part of an international scientific collaboration with Antoine van Oijen (University of Wollongong, Australia) and Michael Cox (University of Wisconsin-Madison), we investigated the sub cellular localization of the E.coli RecA protein which orchestrates the cellular response to DNA damage via its multiple roles in the bacterial SOS response. To do so, Antoine van Oijens group developed a monomeric C-terminal fragment of the lambda repressor as a novel fluorescent probe that specifically interacts with RecA filaments on single-stranded DNA (RecA*). Single-molecule imaging techniques in live cells revealed that RecA is largely sequestered in storage structures during normal metabolism. Upon DNA damage, the storage structures dissolve and the cytosolic pool of RecA rapidly nucleates to form early SOS-signaling complexes, maturing into DNA-bound RecA bundles at later time points. Both before and after SOS induction, RecA* largely appears at locations distal from replisomes. Interestingly, upon completion of DNA repair, RecA storage structures reform. In a collaboration with Myron Goodman (University of Southern California) and Michael Cox (University of Wisconsin-Madison), we investigated how E.coli DNA polymerase V (polV, is regulated by a conformation change upon interacting with ATP and the RecA protein. Mutagenic translesion DNA polymerase V (UmuD'C) is induced as part of the DNA damage-induced SOS response in Escherichia coli, and is subjected to multiple levels of regulation. The UmuC subunit is sequestered on the cell membrane (spatial regulation) and enters the cytosol after forming a UmuD'C complex, 45 min post-SOS induction (temporal regulation). However, DNA binding and synthesis cannot occur until pol V interacts with a RecA nucleoprotein filament (RecA*) and ATP to form a mutasome complex, pol V Mut = UmuD'C-RecA-ATP. The location of RecA relative to UmuC determines whether pol V Mut is catalytically on or off (conformational regulation). In our collaborative study, we reported that binding of ATP triggers a conformational switch that reorients RecA relative to UmuC to activate pol V Mut. This process is required for polymerase-DNA binding and synthesis. We found that Pol V Mut retains RecA in both activated and deactivated states but binding to a primer-template (p/t) DNA occurred only when it is activated. In collaboration with Martin Gonzalez (Southwestern University, TX), we investigated the regulation of the rumAB operon that is found on the integrating conjugative element ICE391 (formerly known as IncJ R391). rumAB encodes for encodes polVICE391, a highly mutagenic homolog of E. coli polV and was initially identified and reported in the literature by scientists in the SDRRM in 1993. polV and its orthologs have previously been shown to be major contributors to spontaneous and DNA damage-induced mutagenesis in vivo. As a result, multiple levels of regulation are imposed on the polymerases so as to avoid aberrant mutagenesis. We discovered that the mutagenesis-promoting activity of polV_ICE391 is additionally regulated by a transcriptional repressor encoded by SetR_ICE391, since Escherichia coli expressing SetR demonstrated reduced levels of polV_ICE391-mediated spontaneous mutagenesis relative to cells lacking SetR. SetR regulation was shown to be specific for the rumAB operon and in vitro studies with highly purified SetR revealed that under alkaline conditions, as well as in the presence of activated RecA, SetR undergoes a self-mediated cleavage reaction that inactivates repressor functions. Electrophoretic mobility shift assays revealed that SetR acts as a transcriptional repressor by binding to a site overlapping the -35 region of the rumAB operon promoter. Our study therefore provided evidence indicating that SetR acts as a transcriptional repressor of the ICE391-encoded mutagenic response. In a collaboration with Anders Clausen (University of Gothenburg, Sweden) we investigated mechanisms of Ribonucleotide Excision Repair (RER) in Saccharomyces cerevisiae. To do so, we utilized a steric gate mutant of DNA polymerase eta that we previously generated in the SDRRM in 2015. pol eta is best known for its ability to bypass UV-induced thymine-thymine (T-T) dimers and other bulky DNA lesions, but it also has other cellular roles. In our study, we presented evidence that pol eta competes with DNA polymerases alpha and delta for the synthesis of the lagging strand genome-wide, where it also shows a preference for T-T in the DNA template. Moreover, we found that the C-terminus of pol eta, which contains a PCNA-Interacting Protein motif is required for pol eta to function in lagging strand synthesis. Our findings provided insight into the physiological role of DNA synthesis by pol eta and have implications for our understanding of how our genome is replicated to avoid mutagenesis, genome instability and cancer. In another international scientific collaboration with Justyna McIntyre at the Polish Academy of Sciences (Warsaw, Poland), we investigated the regulation of human Y-family DNA polymerase iota, which was discovered by scientists in the SDRRM twenty-one years ago. Like all other Y-family polymerases, pol iota interacts with proliferating cell nuclear antigen (PCNA), Rev1, ubiquitin and ubiquitinated-PCNA and is also ubiquitinated itself. In our collaborative study, we reported that pol iota also interacts with the p300 acetyltransferase and is acetylated. The primary acetylation site is K550, located in the Rev1-interacting region. However, K550 amino acid substitutions have no effect on pol iota's ability to interact with Rev1. Interestingly, we found that acetylation of pol iota significantly and specifically increased in response to SN2 alkylating agents and to a lower extent to SN1 alkylating and oxidative agents. Finally, in a collaborative study with Philipp Holligers group at the Medical Research Council in Cambridge, England), we investigated the physicochemical properties of nucleic acids, which are dominated by their highly charged phosphodiester backbone chemistry. The polyelectrolyte structure decouples information content (base sequence) from bulk properties, such as solubility, and has been proposed as a defining trait of all informational polymers. However, this conjecture has not previously been tested experimentally. In our collaborative study, we described the encoded synthesis of a genetic polymer with an uncharged backbone chemistry: alkyl phosphonate nucleic acids (phNAs) in which the canonical, negatively charged phosphodiester was replaced by an uncharged P-alkyl phosphonodiester backbone. Using synthetic chemistry and polymerase engineering, we described the enzymatic, DNA-templated synthesis of P-methyl and P-ethyl phNAs, and the directed evolution of specific streptavidin-binding phNA aptamer ligands directly from random-sequence mixed P-methyl/P-ethyl phNA repertoires. Our results established an example of the DNA-templated enzymatic synthesis and evolution of an uncharged genetic polymer and provided a foundational methodology for their exploration as a source of novel functional molecules.
| 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 |
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