STUDIES ON DNA REPLICATION, REPAIR, AND MUTAGENESIS IN EUKARYOTIC AND PROKARYOTIC CELLS

Roger Woodgate, PhD, Head, Section on DNA Replication, Repair and Mutagenesis
Ekaterina Chumakov, PhD, Staff Scientist
Alexandra Vaisman, PhD, Senior Research Fellow
Mary McLenigan, BS, Chemist
John McDonald, PhD, Biologist
Elena Curti, PhD, Visiting Fellow
Kiyonobu Karata, PhD, Visiting Fellow
Wojciech Kuban, PhD, Visiting Fellow
Eiji Ohashi, PhD, Visiting Fellow
Tara Howard, BSc, Predoctoral Fellow
Padmasini Venkatachari, BSc, Predoctoral Fellow

Section web site

Under optimal conditions, the fidelity of DNA replication is extremely high. Indeed, estimates suggest that, on average, only one error occurs per every 1010 bases replicated. However, as living organisms are continually subjected to a variety of endogenous and exogenous DNA-damaging agents, optimal conditions rarely prevail in vivo. Although all organisms have evolved elaborate repair pathways to deal with such damage, the repair pathways rarely operate with 100 percent efficiency. As a consequence, persisting DNA lesions are replicated, but with much lower fidelity than undamaged DNA. The general aim of our work is to understand the molecular mechanisms by which mutations are introduced into damaged DNA. The process is commonly referred to as translesion DNA synthesis (TLS) or translesion replication (TR) and is facilitated by one or more "Y-family" DNA polymerases, which are conserved from bacteria to humans. Indeed, based on phylogenetic relationships, Y-family polymerases can be broadly classified into five subfamilies. DinB-like (polIV/pol kappa–like) proteins are ubiquitous; the Rev1-like, Rad30A (pol eta)-like, and Rad30B (pol iota)-like polymerases are found only in eukaryotes; UmuC (polV)-like polymerases are found exclusively in prokaryotes. We continue to investigate TLS in all three kingdoms of life: bacteria, archaea, and eukaryotes.

Translesion replication in prokaryotes

Curti, Howard, Karata, Vaisman, Venkatachari, Woodgate

Y-family DNA polymerases are notoriously error-prone when synthesizing undamaged DNA, often leading to increased rates of spontaneous mutagenesis. We recently reported the characterization of polV_R391, a novel prokaryotic Y-family polymerase from the incJ conjugative transposon R391, which has a far superior ability to promote spontaneous mutagenesis than any polV orthologue reported to date. PolV_R391 is encoded by rumA′B and, when expressed in a delta umuDC lexA(Def) recA730 strain, promotes levels of spontaneous mutagenesis that were about 3- or 13-fold greater than do MucA¢B (polR1) or UmuD¢C (polV), respectively. We were therefore interested in determining the mutagenic spectra of RumA¢B-dependent mutations in the hope that the spectra would provide clues to the molecular basis for the enhanced spontaneous mutator activity of the polV-like polymerase. To that end, we used a mutagenesis assay involving E. coli rpoB. The rpoB gene encodes the beta-subunit of RNA polymerase, and mutations in the gene are either lethal or result in Rifampicin resistance. Of the mutations that lead to Rifampicin resistance, 88 percent are concentrated in a region of about 200 bp in the central part of the gene.

Using this approach, we analyzed 681 spontaneous chromosomal rpoB mutants. Of these, 346 were derived from the delta umuDC, lexA(Def) recA730 mutL strain expressing the low copy-number vector pGB2 while 335 were derived from the isogenic strain expressing RumA¢B. The spontaneous mutations derived from the Rum− background concentrated at 14 sites; the spectrum was dominated by eight hotspots (defined as bases with 10 or more mutations), with two of the most significant hotspots at positions 1534 and 1547 of rpoB and comprising 24 and 29 percent of all mutations, respectively. Ninety-eight percent of the recovered mutations were transitions, with A:T→G:C predominating. The expression of RumA¢B coincided with both the appearance of 25 new mutational sites and a marked difference in mutation spectrum. In particular, we observed a dramatic increase in transversions. Indeed, C:G→A:T transversions detected at nine sites accounted for 28 percent of the total base substitutions while A:T→T:A transversions at six sites accounted for 16 percent of mutagenic events. Even though rumAB-dependent replication significantly increased random mutagenesis throughout the 200 bp region of the analyzed rpoB, the spectrum was dominated by 13 distinct hotspots; of these, transversions at nine positions were clearly RumA¢B-dependent as they were absent in the Rum− control strain.

Taken together, our observations indicate that Rum¢B can compete efficiently with E. coli’s resident polymerases for access to genomic DNA; in so doing, it leaves its unique "genetic fingerprint" on the spectrum of rpoB mutations. Biochemical characterization of polV_R391 revealed the enzyme’s exceptional ability to misincorporate T opposite C and T in sequence contexts corresponding to mutagenic hotspots. Purified polV_R391 also traversed a T–T pyrimidine dimer efficiently and displayed greater accuracy opposite the 3¢T of the dimer than opposite an undamaged T. Our study therefore explained the molecular basis for the superior spontaneous mutator activity of RumA¢B and its ability to promote efficient and accurate bypass of T–T pyrimidine dimers in vivo.

Mead S, Vaisman A, Valjavec-Gratian M, Karata K, Vandewiele D, Woodgate R. Characterization of polVR₃₉₁: a Y-family polymerase encoded by rumA′B from the IncJ conjugative transposon, R391. Mol Microbiol 2007;63:797-810.

Schlacher K, Cox MM, Woodgate R, Goodman MF. RecA acts in trans to allow replication of damaged DNA by DNA polymerase V. Nature 2006;442:883-7.

Schlacher K, Jiang Q, Woodgate R, Goodman MF. Purification and characterization of Escherichia coli DNA polymerase V. Methods Enzymol 2006;408:378-90.

Characterization of novel thermostable translesion polymerases from archaea

McDonald, Vaisman, Woodgate; in collaboration with Cadet, Holliger

In the absence of repair, lesions accumulate in DNA. Thus, DNA persisting in specimens of paleontological, archaeological, or forensic interest is inevitably damaged. For many years, Taq polymerase has served as the stalwart enzyme in the PCR amplification of DNA. However, a major limitation of Taq is its inability to amplify damaged DNA, thereby restricting its usefulness in forensic applications and in the amplification of ancient DNA. In a collaborative project with Philipp Holliger, we recently described a strategy for the recovery of genetic information from damaged DNA. In particular, we used the technique of compartmentalized self-replication to generate hybrid polymerases consisting of Thermus aquaticus, Thermus thermophilus, and Thermus flavus DNA polymerases. We selected the novel polymerases for their ability to extend single, double, and even quadruple mismatches; to process noncanonical primer–template duplexes; and to bypass lesions found in ancient DNA, such as hydantoins and abasic sites. When applied to the PCR amplification of 47,000- to 60,000-year-old cave bear DNA, the novel polymerases outperformed wild-type Taq DNA polymerase by up to 150 percent and yielded amplification products at sample dilutions at which Taq no longer yielded a product. Our results demonstrated that engineered polymerases can expand the recovery of genetic information from Pleistocene specimens and may therefore benefit genetic analysis in paleontology, archaeology, and forensic medicine.

D’Abbadie M, Hofreiter M, Vaisman A, Loakes D, Gasparutto D, Cadet J, Woodgate R, Paabo S, Holliger P. Molecular breeding of polymerases for amplification of ancient DNA. Nat Biotechnol 2007;25:939-43.

McDonald JP, Hall A, Gasparutto D, Cadet J, Ballantyne J, Woodgate R. Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs. Nucleic Acids Res 2006;34:1102-11.

Characterization of human DNA polymerases iota

Chumakov, Kuban, Ohashi, Woodgate; in collaboration with Yang

Humans possess four Y-family polymerases: pol eta, pol iota, pol kappa, and Rev1. Like all known polymerases, they require a divalent cation as an activator for phosphatidyl transfer. Two metal ions are usually coordinated by three acidic amino acids within the active site of the polymerase forming a metal bridge between the enzyme and the terminal phoshoryl group of the substrate and facilitating the departure of the pyrophosphate moiety. Based on its cellular abundance, Mg²⁺ is generally believed to be the activating cofactor for DNA polymerases in vivo. However, under certain conditions in vitro, Mn²⁺, Co²⁺, Ni²⁺, and Zn²⁺ have the capacity to substitute for Mg²⁺, but usually with the consequence of reduced fidelity and decreased processivity.

In the past year, we determined the effects of Mn²⁺ and Mg²⁺ on the enzymatic properties of human DNA polymerase iota in vitro. In contrast to other polymerases, pol iota exhibited the greatest activity in the presence of low levels of Mn²⁺ (0.05–0.25mM), with peak activity observed in the presence of Mg²⁺ in the range of 0.1–0.5mM and significant reduction in activity at Mg²⁺ concentrations greater than 2mM. Steady-state kinetic analyses revealed that Mn²⁺ increased the catalytic activity of pol iota between 30-fold and 60,000-fold through a dramatic decrease in the K_m for nucleotide incorporation. Interestingly, while pol iota preferentially misinserted G opposite T by a factor of about 1.4- to 2.5-fold over the correct base A in the presence of 0.25 and 5 mM Mg²⁺, respectively, the correct insertion of A was actually favored two-fold over the misincorporation of G in the presence of 0.075 mM Mn²⁺. Low levels of Mn²⁺ also dramatically increased the ability of pol iota to traverse a cyclobutane pyrimidine dimer, an abasic site, and a benzo[a]pyrene adduct in vitro. Furthermore, titration experiments revealed a strong preference of pol iota for Mn²⁺ even when Mg²⁺ was present in a greater-than-10-fold excess. Our observations therefore raised the intriguing possibility that the cation used by pol iota in vivo may be Mn²⁺ rather than, as generally assumed, Mg²⁺.

Frank EG, Woodgate R. Increased catalytic activity and altered fidelity of human DNA polymerase iota in the presence of manganese. J Biol Chem 2007;282:24689-96.

Martomo SA, Yang WW, Vaisman A, Maas A, Yokoi M, Hoeijmakers JH, Hanaoka F, Woodgate R, Gearhart PJ. Normal hypermutation in antibody genes from congenic mice defective for DNA polymerase iota. DNA Repair 2006;5:392-8.

Plosky BS, Vidal AE, Fernández de Henestrosa AR, McLenigan MP, McDonald JP, Mead S, Woodgate R. Controlling the subcellular localization of DNA polymerases iota and eta via interactions with ubiquitin. EMBO J 2006;25:2847-55.

Yang W, Woodgate R. What a difference a decade makes: insights into translesion DNA synthesis. Proc Natl Acad Sci USA 2007;104:15591-8.

Role of DNA polymerase iota in humans with the xeroderma pigmentosum variant (XP-V) syndrome

McLenigan, Vaisman, Woodgate; in collaboration with Maher

Xeroderma pigmentosum variant (XPV) patients have normal DNA excision repair but are predisposed to developing sunlight-induced cancer. They exhibit a 20-fold-higher-than-normal frequency of UV-induced mutations and a highly unusual spectrum of mutations. The primary defect in XP-V cells is a lack of functional pol eta, which normally inserts adenine nucleotides opposite photoproducts involving thymine. The high frequency and striking difference in the spectrum of UV-induced mutations in XP-V cells strongly suggest that, in the absence of pol eta, translesion replication of UV-induced DNA lesions is catalyzed by an alternate DNA polymerase that is much more error-prone than pol eta. One such candidate enzyme is pol iota. Indeed, our previous replication assays demonstrated that pol iota replicates past 5¢T-T3¢ and 5¢T-U3¢ cyclobutane pyrimidine dimers, incorporating G or T nucleotides opposite the 3¢ nucleotide. In a collaborative study with Veronica Maher, we transfected an infinite lifespan XP-V cell line with antisense to POLI to test the hypothesis that pol iota causes the high frequency and abnormal spectrum of UV-induced mutations in XP-V cells. We subsequently identified two stable cell lines in which pol iota expression was halved while the spectrum of mutations was the same as the parental XP-V cell line. Our data therefore strongly support the hypothesis that, in cells lacking pol eta, pol iota is responsible for the high frequency and abnormal spectrum of UV-induced mutations and, ultimately, their malignant transformation.

Vaisman A, Takasawa K, Iwai S, Woodgate R. DNA polymerase iota-dependent translesion replication of uracil containing cyclobutane pyrimidine dimers. DNA Repair 2006;5:210-8.

Wang Y, Woodgate R, McManus TP, Mead S, McCormick JJ, Maher VM. Evidence that in xeroderma pigmentosum variant cells, which lack DNA polymerase eta, DNA polymerase iota causes the very high frequency and unique spectrum of UV-induced mutations. Cancer Res 2007;67:3018-26.

COLLABORATORS

Jean Cadet, PhD, Laboratoire Lésions des Acides Nucléiques, CEA-Grenoble, Grenoble, France
Philipp Holliger, PhD, Medical Research Council, Cambridge, UK
Veronica Maher, PhD, Michigan State University, East Lansing, MI
Wei Yang, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD

For further information, contact woodgate@helix.nih.gov.

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