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Studies on DNA Replication, Repair, and Mutagenesis in Eukaryotic and Prokaryotic Cells
- Roger Woodgate, PhD, Head, Laboratory of Genomic Integrity
- Ekaterina Chumakov, PhD, Staff Scientist
- Alexandra Vaisman, PhD, Senior Research Fellow
- John McDonald, PhD, Biologist
- Mary McLenigan, BS, Chemist
- Wojciech Kuban, PhD, Visiting Fellow
- Justyna McIntyre, PhD, Visiting Fellow
- Katherine Donigan, PhD, Intramural Research Training Award Fellow
- Sender Lkhagvadorj, PhD, Intramural Research Training Award Fellow
- Donald Huston, Student Fellow
- 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 (1).
Novel expression vectors for the simple and efficient purification of E.coli pol V and human DNA polymerases eta, iota, and nu
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 (2). 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, facilitating the correct folding of the recombinant protein and increasing 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 we showed that the purified DNA polymerases are catalytically active in vitro (3).
Characterization of E. coli pol V
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. The strong activity was attributed to unexpectedly high processivity of pol V Mut (a complex that formed in vitro consisting of UmuD'2C-RecA-ATP), which was recruited to a primer terminus by Single-stranded binding protein (2). Remarkably, under these conditions, wild-type pol V Mut efficiently incorporated ribonucleosides into DNA (4). 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 (4). 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 the 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 did 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) are attributable to excessive incorporation of rNTPs during TLS that are subsequently targeted for repair, rather than to inability to traverse UV-induced lesions.
Converting a DNA polymerase into an RNA polymerase
In a collaboration with Philip Holliger, we further investigated DNA polymerase substrate specificity, which is fundamental to genome integrity and to polymerase applications in biotechnology (5). We reported the discovery of a novel specificity checkpoint located over 25 Ǻ 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.
- Sale JE, Lehmann AR, Woodgate R. Y-family DNA polymerases and their role in tolerance of cellular DNA damage. Nat Rev Mol Cell Biol 2012;13:141-152.
- Karata K, Vaisman A, Goodman MF, Woodgate R. Simple and efficient purification of Escherichia coli DNA polymerase V: cofactor requirements for optimal activity and processivity in vitro. DNA Repair 2012;11:431-440.
- Frank EG, McDonald JP, Karata K, Huston D, Woodgate R. A strategy for the expression of recombinant proteins traditionally hard to purify. Anal Biochem 2012;429:132-139.
- 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.
- Cozens C, Pinheiro VB, Vaisman A, Woodgate R, Holliger P. A short adaptive path from DNA to RNA polymerases. Proc Natl Acad Sci USA 2012;109:8067-8072.
- 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