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 (polV). The polymerase lacks 3' to 5' exonucleolytic proofreading activity and is inherently error-prone when replicating both undamaged and damage DNA. So as to limit any gratuitous mutagenesis, the activity of polV is strictly regulated in the cell at multiple levels. The first is transcriptional control. Like all genes expressed as part of the damage-inducible SOS response, the umuDC locus is negatively-regulated by the LexA transcriptional repressor, which binds tightly to a near consensus binding-site immediately upstream of the umuDC operon. As a consequence, the UmuDC proteins are induced late in the SOS response. Indeed, they are not fully derepressed until 15 minutes after cells have been exposed to DNA damage and significant levels of the Umu proteins do not accumulate until 45 minutes after DNA damage. Levels of UmuD and UmuC are further kept to a minimum through their targeted proteolysis by the ATP-dependent protease, Lon. Even when expressed, the homodimeric UmuD protein is considered to be mutagenically inactive and has to undergo a RecA-mediated self-cleavage reaction that removes its N-terminal 24 amino acids, thereby converting UmuD into the mutagenically active UmuD' protein. This cleavage reaction is inefficient and results in the formation of heterodimeric UmuD/UmuD'. Within the context of the heterodimer, UmuD' is subject to rapid proteolysis by the ClpXP protease, such that homodimeric UmuD'2 only accumulates in the presence of continued DNA damage. As a result, formation of UmuD'2C (polV) only occurs in the presence of persistent cellular DNA damage. Biochemical studies have revealed that polV has intrinsically weak catalytic activity, but this activity is dramatically stimulated by interactions with ATP and RecA to form a higher order complex termed polV Mut. The activity of polV Mut is further enhanced through protein-protein interactions with the beta-sliding clamp and Single-Stranded Binding (SSB) protein. It is evident that numerous and complex levels of regulation have therefore been imposed on polV, so as to apparently limit its highly mutagenic functions within the cell. However, a low level of mutagenesis is actually beneficial, since it provides genetic diversity and may contribute to overall evolutionary fitness. Indeed, E.coli appears to utilize the various polV regulatory pathways to provide just the right amount of polV in times of stress, so as to help the organism overcome environmentally challenging adversity. Our studies on the eukaryotic TLS polymerases focused on human DNA polymerases (pols) eta and iota. Both are Y-family DNA polymerase paralogs that facilitate translesion synthesis (TLS) past damaged DNA. Like E.coli polV, the activity of pols eta and iota can be modulated through posttranslational modifications. Indeed, both pol eta and pol iota can be monoubiquitinated in vivo. Pol eta has previously been shown to be ubiquitinated at one primary site. When this site is unavailable, three nearby lysines, may become ubiquitinated. In contrast, mass spectrometry analysis of monoubiquitinated pol iota revealed that it is ubiquitinated at over 27 unique sites. Many of these sites are localized in different functional domains of the protein, including the catalytic polymerase domain, the PCNA-interacting region, the Rev1-interacting region, as well as its Ubiquitin Binding Motifs, UBM1 and UBM2. Pol iota monoubiquitination remained unchanged after cells were exposed to DNA damaging agents such as UV-light (generating UV-photoproducts), ethyl methanesulfonate (generating alkylation damage), mitomycin C (generating interstrand crosslinks), or potassium bromate (generating direct oxidative DNA damage). However, when exposed to naphthoquinones, such as menadione and plumbagin, which cause indirect oxidative damage through mitochondrial dysfunction, pol iota becomes transiently polyubiquitinated via K11- and K48-linked chains of ubiquitin and subsequently targeted for degradation. Polyubiquitination does not occur as a direct result of the perturbation of the redox cycle, as no polyubiquitination was observed after treatment with rotenone, or antimycin A, which inhibit mitochondrial electron transport. Interestingly, polyubiquitination was observed after the inhibition of the lysine acetyltransferase, KATB3/p300. We hypothesized that the formation of polyubiquitination chains attached to pol iota occurs via the interplay between lysine acetylation and ubiquitination of ubiquitin itself at K11- and K48- rather than oxidative damage per se. As part of a collaborative study with Patricia Gearhart (NIA), we also investigated the role that pol iota plays in the somatic hypermutation of antibody genes. Pol iota is an attractive candidate for somatic hypermutation in antibody genes because of its low fidelity. To identify a role for pol iota, we analyzed mutations in two strains of mice with deficiencies in the enzyme: 129X1/SvJ mice with negligible expression of truncated pol iota, and knock-in mice that express full-length pol iota that is catalytically inactive. Both strains had normal frequencies and spectra of mutations in the variable region, indicating that loss of pol iota did not change overall mutagenesis. We next examined if pol iota affected tandem mutations generated by another error-prone polymerase, pol zeta. The frequency of contiguous mutations was analyzed using a novel computational model to determine if they occur during a single DNA transaction, or during two independent events. Analyses of 2,000 mutations from both strains indicated that pol iota compromised mice lost the tandem signature, whereas C57BL/6 mice accumulated significant amounts of double mutations. The results therefore support a model where pol iota occasionally accesses the replication fork to generate a first mutation, and pol zeta extends the mismatch with a second mutation.
| 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 |
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| 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 |
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| Vaisman, Alexandra; Woodgate, Roger (2015) Redundancy in ribonucleotide excision repair: Competition, compensation, and cooperation. DNA Repair (Amst) 29:74-82 |
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