Studies on DNA Replication, Repair, and Mutagenesis in Eukaryotic and Prokaryotic Cells
- Roger Woodgate, PhD, Chief, Section on DNA Replication, Repair, and Mutagenesis
- Ekaterina Chumakov, PhD, Staff Scientist
- Alexandra Vaisman, PhD, Interdisciplinary Scientist
- John McDonald, PhD, Biologist
- Mary McLenigan, BS, Chemist
- Ewa Grabowska, PhD, Postdoctoral Visiting Fellow
- In Soo Jung, PhD, Postdoctoral Visiting Fellow
- Ashley Swanson, PhD, Postdoctoral Intramural Research Training Award Fellow
- Erin Walsh, PhD, Postdoctoral Intramural Research Training Award Fellow
- Donald Huston, BS, Technical Intramural Research Training Award Trainee
- David Wilson, BS, Postbaccalaureate Intramural Research Training Award Fellow
Under optimal conditions, the fidelity of DNA replication is extremely high. Indeed, it is estimated that, on average, only one error occurs for every 10 billion bases replicated. However, given that living organisms are continually subjected to a variety of endogenous and exogenous DNA–damaging agents, optimal conditions rarely prevail in vivo. While all organisms have evolved elaborate repair pathways to deal with such damage, the pathways rarely operate with 100% efficiency. Thus, persisting DNA lesions are replicated, but with much lower fidelity than in undamaged DNA. Our aim is to understand the molecular mechanisms by which mutations are introduced into damaged DNA. The process, commonly referred to as translesion DNA synthesis (TLS), is facilitated by one or more members of the Y-family of DNA polymerases, which are conserved from bacteria to humans. Based on phylogenetic relationships, Y-family polymerases may be broadly classified into five subfamilies: DinB-like (polIV/pol kappa–like) proteins are ubiquitous and found in all domains of life; in contrast, the Rev1-like, Rad30A (pol eta)–like, and Rad30B (pol iota)–like polymerases are found only in eukaryotes; and the UmuC (polV)–like polymerases only in prokaryotes. We continue to investigate TLS in all three domains of life: bacteria, archaea, and eukaryotes.
Prokaryotic DNA repair and mutagenesis
Most damage-induced mutagenesis in Escherichia coli is dependent upon the Umu′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 (Reference 1, 2). 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 to a near-consensus binding site immediately upstream of the operon. As a consequence, the UmuDC proteins are induced late in the SOS response. Indeed, they are not fully derepressed until about 15 minutes after cells have been exposed to DNA damage, and significant levels of the Umu proteins do not accumulate until approximately 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. The 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′ 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 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 beneficial, because 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.
Eukaryotic DNA repair and mutagenesis
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 post-translational 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 (Reference 3). Many of these sites are located 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, given that 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, we also investigated the role that pol iota plays in the somatic hypermutation of antibody genes (Reference 4). 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 whether pol iota affected tandem mutations generated by another error-prone polymerase, pol zeta. We analyzed the frequency of contiguous mutations using a novel computational model to determine whether 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 in which pol iota occasionally accesses the replication fork to generate a first mutation, and pol zeta extends the mismatch with a second mutation.
Additional Funding
- Collaborative extramural U01 grant (U01HD085531-01) with Prof. Digby Warner, University of Cape Town, South Africa: Replisome dynamics in M. tuberculosis: linking persistence to genetic resistance
Publications
- Goodman MF, McDonald JP, Jaszczur MM, Woodgate R. Insights into the complex levels of regulation imposed on Escherichia coli DNA polymerase V. DNA Repair 2016;44:42-50.
- Jaszczur M, Bertram JG, Robinson A, van Oijen AM, Woodgate R, Cox MM, Goodman MF. Mutations for worse or better: low-fidelity DNA synthesis by SOS DNA polymerase V is a tightly regulated double-edged sword. Biochemistry 2016;55:2309-2318.
- McIntyre J, McLenigan MP, Frank EG, Dai X, Yang W, Wang Y, Woodgate R. Posttranslational regulation of human DNA polymerase iota. J Biol Chem 2015;290:27332-27344.
- Maul RW, MacCarthy T, Frank EG, Donigan KA, McLenigan MP, Yang W, Saribasak H, Huston DE, Lange SS, Woodgate R, Gearhart PJ. DNA polymerase iota functions in the generation of tandem mutations during somatic hypermutation of antibody genes. J Exp Med 2016;213:1675-1683.
Collaborators
- Michael Cox, PhD, University of Wisconsin, Madison, WI
- Patricia J. Gearhart, PhD, Laboratory of Molecular Biology and Immunology, NIA, Baltimore, MD
- Myron F. Goodman, PhD, University of Southern California, Los Angeles, CA
- Andrew Robinson, PhD, University of Wollongong, Wollongong, Australia
- Anton Simeonov, PhD, Scientific Director, NCATS, Bethesda, MD
- Antoine Van Oijen, PhD, University of Wollongong, Wollongong, Australia
- Yinsheng Wang, PhD, University of California, Riverside, CA
- Digby Warner, PhD, University of Cape Town, Cape Town, South Africa
- Wei Yang, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
Contact
For more information, email woodgate@.nih.gov or visit sdrrm.nichd.nih.gov.
