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Studies on DNA Replication, Repair, and Mutagenesis in Eukaryotic and Prokaryotic Cells

Roger Woodgate, PhD
  • Roger Woodgate, PhD, Head, Laboratory of Genomic Integrity
  • Ekaterina Chumakov, PhD, Staff Scientist
  • Alexandra Vaisman, PhD, Interdisciplinary Scientist
  • John McDonald, PhD, Biologist
  • Mary McLenigan, BS, Chemist
  • Justyna McIntyre, PhD, Visiting Fellow
  • Katherine Donigan, PhD, Intramural Research Training Award Fellow
  • Sender Lkhagvadorj, PhD, Intramural Research Training Award Fellow
  • Donald Huston, BS, Technical Intramural Research Training Award Trainee
  • Gregory Loeb, Student 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, the 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 that 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.

Mechanisms of ribonucleotide repair in Escherichia coli

Most damage-induced mutagenesis in Escherichia coli is dependent upon the UmuD′C protein complex, which contains DNA polymerase V (pol V) (1). We recently discovered that pol V is also characterized by substantially reduced sugar selectivity. When the canonical Watson-Crick base pairing is preserved, purified pol V, accompanied by accessory proteins, readily incorporates all four ribonucleoside monophosphates (NMPs) and catalyzes efficient and highly processive RNA synthesis in vitro in the presence of all four ribonucleoside triphosphates (rNTPs). The ability of pol V to incorporate ribonucleotides is dramatically enhanced by a Y11A substitution at the conserved steric gate residue of UmuC (2). Interestingly, while UmuC_Y11A is highly inaccurate in vitro, it exhibits low mutability in vivo. Furthermore, despite the observation that the UmuC_Y11A variant catalyzed TLS past a T-T cyclobutane pyrimidine dimer (CPD) in vitro at least as efficiently as the wild-type enzyme, it conferred minimal UV resistance to a delta umuDC strain. To explain these phenotypes, we suggested that the dramatic increase in rNMP incorporation promoted by UmuC_Y11A leads to the induction of downstream pathways involving rNMP processing. Specifically, the rNMP-targeted repair pathways would not only reduce umuC_Y11A–dependent spontaneous and UV–induced mutagenesis, but also interfere with completion of TLS resulting in the observed decrease in UV resistance.

The major enzymes initiating this pathway are the ribonucleotide-specific endonucleases ribonuclease H (RNase H), which are present in organisms across all domains and are classified as types 1 and 2, based on sequence conservation and substrate preference.

Taking advantage of the different capacities for ribonucleotide incorporation by the UmuC_Y11A pol V variant, we examined rNMP–processing pathways that cause phenotypic changes in strains expressing the pol V variants. While there was an approximately four-fold increase in the absolute number of Y11A–dependent mutations in the delta rnhA strain (lacking RNase HI) compared with the rnh+ strain, umuC_Y11A mutagenesis actually decreased from 7–5% of the wild-type levels, when expressed as a percentage of wild-type pol V–dependent mutagenesis. In contrast, in the isogenic delta rnhB strain (lacking RNase HII), the number of umuC_Y11A–dependent revertants increased approximately five-fold compared with the rnhB+ strain and reached about 40% of the level of mutagenesis observed with wild-type pol V (3).

Our studies clearly demonstrated that RNase HII participates in a repair pathway that reduces the accumulation of rNMPs, as well as incorrect deoxyribonucleoside monophosphates (dNMPs) incorporated into undamaged and damaged DNA by UmuC_Y11A.  Based upon its in vitro properties, we expected pol V umuC_Y11A to be as mutagenic as the wild-type pol V, if not more so.  However, even in the delta rnhB strain, Y11A–dependent mutagenesis was less than half of that observed with wild-type pol V, indicating that additional repair pathways act to reduce the mutagenic consequences of rNMPs incorporated by the highly error-prone umuC_Y11A.  Indeed, in the umuC_Y11A of the isogenic delta rnhA delta rnhB strain spontaneous mutagenesis rose significantly to about 72% of the level observed with wild-type pol V. Our studies therefore revealed that although RNase HI alone does not appear to participate in the removal of ribonucleotides incorporated by umuC_Y11A, in the absence of RNase HII, where there is likely to be a significant accumulation of ribonucleotides into DNA, RNase HI helps reduce the mutagenic burden of errant ribonucleotide incorporation into the E. coli genome.

Whilst umuC_Y11A–dependent mutagenesis, relative to wild-type pol V in the delta rnhB delta rnhA strain, was significantly higher than the wild-type strain, it was still less than that promoted by wild-type pol V, despite the fact the enzyme exhibits the same low base selectivity in vitro. These observations indicate that additional mechanisms must exist that target the ribonucleotides incorporated by umuC_Y11A for repair. We therefore took advantage of the pol V phenotype to investigate the contribution of mismatch repair (MMR), base excision repair (BER), and nucleotide excision repair (NER) to ribonucleotide excision repair (RER) (4). We found no evidence for a significant role of either MMR or BER in back-up RER pathways. Somewhat surprisingly, we discovered that umuC_Y11A–dependent mutagenesis increased significantly in delta uvrA, uvrB5, and delta 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 by reconstituting ribonucleotide-dependent NER in vitro. We demonstrated that UvrABC nuclease–catalyzed incisions are readily made on DNA templates containing one, two, or five rNMPs and that the reactions are stimulated by the presence of mispaired bases. Similar to NER of DNA lesions, excision of rNMPs proceeds through dual incisions made at the eighth phosphodiester bond 5′ and fourth–fifth phosphodiester bonds 3′ of the ribonucleotide. Our studies have thus revealed that efficient ribonucleotide repair in E. coli, and most likely other prokaryotes, is achieved though the concerted actions of rnhB, rnhA, and NER.

Ubiquitin mediates the physical and functional interaction between human DNA polymerases iota and eta.

Studies on the human TLS polymerases focused on DNA polymerase eta and iota. Both polymerases (pols) co-localize in “replication factories” in vivo after cells are exposed to UV light, and this co-localization is mediated through a physical interaction between the two TLS pols. The regions responsible for the interaction were previously loosely mapped to the C-terminal 200 or so amino acids of each protein. Although the two polymerases clearly co-localize at sites of DNA damage, the kinetics of their re-localization differs, suggesting that the two polymerases are not tightly associated in a living cell. Our studies are beginning to shed light on how such an interaction is facilitated and regulated (5).

We identified the region in pol eta responsible for the interaction with pol iota as its “ubiquitin-binding zinc finger” (UBZ) motif. Similarly, we demonstrated that the region responsible for the interaction between pol iota and pol eta is pol iota’s “ubiquitin-binding motif” (UBM). Given that pol iota is also known to be monoubiquitinated in vivo, we hypothesized that the preferred partner of pol eta might actually be a ubiquitinated form of pol iota. To test the hypothesis, we generated a chimera in which the N-terminus of ubiquitin was fused to the C-terminus of pol iota. The mutant chimera lacked the two C-terminal glycine residues, and therefore only allowed for non-covalent interactions. The chimera interacted avidly with pol eta in the two-hybrid assays, and this interaction was dependent upon I44 of ubiquitin (in the pol iota-Ub chimera). When expressed in human HEK293T cells, the pol iota-Ub chimera was also able to “pull-down” considerably more pol eta than wild-type pol iota. We therefore concluded that the preferred partner for pol eta is a ubiquitinated form of pol iota.

The functional importance of the pol eta–pol iota interaction was clearly demonstrated by the fact that mutants of pol iota that are unable to interact with pol eta exhibit reduced accumulation into replication factories. Conversely, the pol iota–Ub chimera, which exhibited a tighter interaction with pol eta, exhibited enhanced accumulation into replication foci.

Given the complex set of protein-protein interactions that pol eta and pol iota are known to participate in, it seemed reasonable to predict that the ubiquitination status of the pols allows a cell a variety of ways to regulate the formation of TLS complexes. For example, monoubiquitination of pol eta is known to inhibit an interaction with ubiquitinated PCNA, but we demonstrated that it enhances its interaction with pol iota. Upon DNA damage, pol eta is de-ubiquitinated, leading to reduced ability to interact with pol iota but concomitant increased ability to interact with ubiquitinated PCNA, which helps explain why the polymerases exhibit different sub-cellular mobility in a living cell.

Additional Funding

  • NIH Director’s Challenge Award

Publications

  1. Goodman MF, Woodgate R. Translesion DNA polymerases. Cold Spring Harb Perspect Biol 2013;5(10):a010363 (online).
  2. Vaisman A, Kuban W, McDonald JP, Karata K, Yang W, Goodman MF, Woodgate R. Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity. Nucleic Acids Res 2012;40:6144-6157.
  3. McDonald JP, Vaisman A, Kuban w, Goodman MF, Woodgate R. Mechanisms employed by Escherichia coli to prevent ribonucleotide incorporation into genomic DNA by pol V. PLoS Genet 2012;8:e1003030.
  4. Vaisman A, McDonald JP Huston D, Kuban W, Liu L, Van Houten B, Woodgate R. Removal of misincorporated ribonucleotides from prokaryotic genomes: an unexpected role for nuclelotide excision repair. PLoS Genet 2013;in press.
  5. McIntyre J, Vidal AE, McLenigan MP, Bomar MG, Curti E, McDonald JP, Plosky BS, Ohashi E, Woodgate R. Ubiquitin mediates the physical and functional interaction between human DNA polymerases eta and iota. Nucleic Acids Res 2013;41:1649-1660.

Collaborators

  • Myron F. Goodman, PhD, University of Southern California, Los Angeles, CA
  • Philipp Holliger, PhD, Medical Research Council, Cambridge, United Kingdom
  • Alan Lehmann, PhD, Genome Damage and Stability Centre, University of Sussex, Brighton, UK
  • Julian Sale, MD, PhD, Medical Research Council, Cambridge, UK
  • Wei Yang, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD

Contact

For more information, email woodgate@mail.nih.gov or visit lgi.nichd.nih.gov.

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