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
Samantha Mead, PhD, Visiting Fellow
Brian Plosky, PhD, Postdoctoral Fellow
Emily Dotter, BS, Predoctoral Fellow

Under optimal conditions, the fidelity of DNA replication is extremely high. Indeed, it is estimated that, on average, only one error occurs in 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, these pathways rarely operate with 100 percent efficiency. As a consequence, persisting DNA lesions are replicated, but with much lower fidelity than undamaged DNA. We therefore aim 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 that 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-like (pol eta), and Rad30B-like polymerases (pol iota) are found only in eukaryotes; and, in contrast, UmuC-like polymerases (polV) 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, Dotter, Karata, Mead, Vaisman, Woodgate; in collaboration with Goodman

In E. coli, efficient translesion replication of many DNA lesions occurs only when the UmuC protein physically interacts with a dimer of UmuD′ to form a heterotrimeric complex of UmuD′₂C (known as E. coli pol V). In vitro replication assays reveal that regulation of the catalytic activity of pol V is modulated through several protein-protein interactions. For example, pol V does not catalyze TLS alone but instead is an essential component of a multiprotein "mutasome" complex composed of RecA protein, beta sliding-clamp, and SSB. In collaboration with Myron Goodman, we investigated in detail the nature of the interactions between RecA and pol V. It had previously been assumed that RecA binds to the damaged DNA template strand being copied by polV. Remarkably, however, our recent studies revealed that, in the presence or absence of the beta-processivity clamp, polV-catalyzed translesion synthesis occurs only when RecA nucleoprotein filaments assemble on separate single-stranded (ss)DNA molecules in trans. A 3′-proximal RecA filament end-on trans DNA is essential for stimulation and is strengthened by further polV-RecA interactions occurring elsewhere along a trans nucleoprotein filament. Based on these observations, we suggested that, despite the absolute requirement of RecA for SOS mutagenesis, trans-stimulation of polV by RecA bound to ssDNA reflects a distinctive regulatory mechanism of mutation that resolves the paradox of RecA filaments assembled in cis on a damaged template strand obstructing translesion DNA synthesis.

Y-family DNA polymerases are notoriously error-prone when synthesizing undamaged DNA. Such mutator activity often results in increased levels of spontaneous mutagenesis. We recently reported the characterization of polVR391, a novel prokaryotic Y-family polymerase from the incJ conjugative transposon R391. The polymerase has a far superior ability to promote spontaneous mutagenesis than any polV orthologue reported to date, is encoded by rumA¿B and, when expressed in a ΔumuDC lexA(Def) recA730 strain, promotes levels of spontaneous mutagenesis that are about three- to 13-fold greater than MucA′B (polR1) or UmuD′C (polV), respectively. Analysis of the spectrum of polVR391-dependent mutations in rpoB revealed a unique "genetic fingerprint" that is typified by an increase in C:G→A:T and A:T&rarrT:A transversions at certain mutagenic hot spots. Biochemical characterization of polVR391 revealed the enzyme's exceptional ability to misincorporate T opposite C and T in sequence contexts corresponding to mutagenic hot spots. Purified polVR391 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.

Identification and characterization of novel Dop4-like enzymes in archaea

McDonald, Woodgate; in collaboration with Ballantyne, Cadet

The ability to detect DNA polymorphisms with molecular genetic techniques has revolutionized the forensic analysis of biological evidence. DNA typing now plays a critical role in the criminal justice system, but one of the technology's limiting factors is that DNA isolated from biological stains recovered from a crime scene is sometimes so damaged as to be intractable to analysis. 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. In contrast, Y-family DNA polymerases, such as Dpo4 from Sulfolobus solfataricus, can traverse a wide variety of DNA lesions. While Dpo4 can perform PCR at moderately high temperatures, it is not as thermostable or processive as Taq polymerase. We were therefore interested in identifying novel Dpo4-like enzymes that might possess greater processivity or thermostability than Dpo4. To do so, we used the amino acid sequences of Dpo4 proteins encoded by S. acidocaldarius Dbh (Sa), S. solfataricus Dpo4 (Sso), and S. tokodaii to design degenerate PCR primers that we subsequently used to PCR-amplify additional Dpo4-like genes from various strains of Sulfolobaceae that grow at temperatures ranging from 75-90°C. We were successful in identifying five new polIV homologues from Acidianus infernus (Ai), Sulfolobus shibatae (Ssh), Sulfolobus tengchongensis (Ste), Stygiolobus azoricus (Saz), and Sulfurisphaera ohwakuensis (Soh). To characterize the biochemical properties of the new polIV-like enzymes, we overexpressed the enzymes in E. coli and purified them by using a similar but somewhat simpler scheme than that previously reported for Dpo4. Such an approach allowed us to purify readily anywhere between 1 and 5 mg of each polymerase from just 1-2 L of E. coli cell culture. Once we had purified the polymerases to greater than 95 percent homogeneity, we characterized them for their relative degree of processivity and lesion-bypassing activities. Of the five novel enzymes, Ste Dpo4 was the most robust and appeared to be as good as, if not better than, Dpo4 at high-temperature, processive DNA synthesis. The Ai-Dpo4 enzyme was not as processive, but, by interchanging its "Little Finger" domain with that of Sso-Dpo4 or Ste-Dpo4, we increased its processivity considerably such that the Ai/Sso and Ai/Ste Dpo4-like chimeras were the most efficient of the novel enzymes at bypassing a variety of DNA lesions.

We irradiated human genomic DNA with UVC light and attempted to amplify Alu short interspersed elements. Taq alone gave an Alu signal that was barely detectable. Remarkably, we obtained a 40-fold increase in the amplicon when we used a blend of Taq with Sso Dpo4 in the PCR reaction. Our findings therefore have immediate and obvious applications in both forensic science and the analysis of ancient DNA samples, which are often degraded and not easily amplified by Taq alone.

Characterization of human DNA polymerases iota and eta

Chumakov, McDonald, McLenigan, Mead, Plosky, Vaisman, Woodgate; in collaboration with Gearhart, West

Humans possess four Y-family polymerases—pol eta, pol iota, pol kappa, and Rev1—that all have spacious active sites capable of accommodating a wide variety of geometric distortions. As a consequence, these polymerases are considerably more error-prone than high-fidelity replicases. Therefore, it is hardly surprising that the in vivo activity of these polymerases is tightly regulated so as to minimize their inadvertent access to primer termini. We recently reported that one such mechanism employed by human cells relies on a specific and direct interaction between DNA polymerases iota and eta with ubiquitin (Ub). Indeed, we showed that both polymerases interact noncovalently with free polyUb chains as well as with mono-ubiquitinated proliferating cell nuclear antigen (Ub-PCNA). We isolated mutants of pol iota (P692R) and pol eta (H654A) that were defective in their interactions with polyUb and Ub-PCNA but that retained their ability to interact with unmodified PCNA. Interestingly, the polymerase mutants exhibited significantly lower levels of replication foci in response to DNA damage, thereby highlighting the biological importance of the polymerase-Ub interaction in regulating access of the TLS polymerases to stalled replication forks in vivo.

Unfortunately, despite possessing efficient mechanisms for targeting TLS enzymes to sites of DNA damage, a stalled fork may nevertheless collapse and undergo repair by homologous recombination. In collaboration with Stephen West, we used fractionated cell-free extracts and purified recombinant proteins to demonstrate that pol eta binds to DNA and extends DNA synthesis from D-loop recombination intermediates in which an invading strand serves as the primer for DNA extension. Extracts prepared from human XP-V cells, defective for pol eta, exhibited severely reduced D-loop extension activity. The ability of pol eta to promote D-loop extension is unusual, as such a reaction could not be promoted by the replicative DNA polymerase delta or by pol iota. We also found that pol eta interacts with the RAD51 recombinase and that pol eta-mediated D-loop extension activity is stimulated by the presence of RAD51. Our results therefore suggest a novel dual function for pol eta at stalled replication forks as well as in the promotion of translesion synthesis and in the reinitiation of DNA synthesis by homologous recombination repair.

Previous studies have shown that several low-fidelity DNA polymerases participate in generating mutations in immunoglobulin genes. DNA pol eta is clearly involved in the process by causing substitutions of A:T base pairs, whereas polymerase iota plays a controversial role. Although the frequency of mutations decreased in the BL2 cell line deficient for Poli, hypermutation was normal in the 129 strain of mice, which carry a natural nonsense mutation in their Poli gene. It is therefore a formal possibility that the 129-derived strains of mice compensated for the Poli defect over time, possibly by using pol eta as a substitute. To examine the role of pol iota in the somatic mutation of immunoglobulin genes in a genetically defined background, we backcrossed the 129-derived Poli nonsense mutation to the C57BL/6 strain for six generations. We studied class-switch recombination and hypermutation in these mice as well as in congenic mice doubly deficient for both polymerases iota and eta. The absence of both polymerases did not affect production of IgG1, indicating that the enzymes are not involved in switch recombination. Furthermore, Poli(-/F6) mice had the same types of nucleotide substitution in variable genes as their C57BL/6 counterparts, and mice doubly deficient for polymerases iota and eta had the same mutational spectrum as Polh/- mice. Thus, our studies confirmed that pol iota does not contribute to the mutational spectra of immunoglobulin genes even in the absence of pol eta.

Role of DNA polymerase iota in humans with Xeroderma Pigmentosum Variant (XP-V) syndrome

Mead, Vaisman, Woodgate; in collaboration Iwai, Maher

Analysis of the spectrum of UV-induced mutations generated in synchronized wild-type S-phase cells reveals that only about 25 percent of mutations occur at thymine (T) while 75 percent are targeted to cytosine (C). The mutational spectra changes dramatically in XP-V cells, which are devoid of pol eta and where about 45 percent of mutations occur at Ts and about 55 percent at Cs. At present, it is unclear whether the C→T mutations represent true misincorporations opposite C or occur perhaps as the result of the correct incorporation of adenine (A) opposite a C in a UV-photoproduct that has undergone deamination to uracil (U). To assess the role, if any, that human pol iota might play in the replicative bypass of such UV-photoproducts, we analyzed the efficiency and fidelity of pol iota-dependent bypass of a T-U cyclobutane pyrimidine dimer (CPD) in vitro. Interestingly, pol iota-dependent bypass of a T-U CPD occurred more efficiently than that of a corresponding T-T CPD. Guanine (G) was misincorporated opposite the 3→U of the T-U CPD only half as frequently as A, the correct Watson-Crick base. While pol iota generally extended the G:3′U-CPD mispairs less efficiently than the correctly paired primer, pol iota-dependent extension was equal to or greater than that observed with human pol eta and pol kappa and S. cerevisiae pol zeta under the same assay conditions.

We believe that the unique pattern of misincorporations that we previously observed during the pol iota-dependent bypass of a T-T CPD and a T-U CPD (described above) readily explains the abnormal spectrum of mutations observed in XP-V cells. For example, pol iota misincorporates G and T opposite the 3→T of the T-T CPD, which would lead to the increase in T→C and T→A substitutions observed. Similarly, pol iota misincorporates T opposite the 3→U of the T-U CPD, which would give rise to the C→A mutations observed in XP-V cells. The decrease in C¿T transitions can also be explained by the propensity of pol iota to "misincorporate" G opposite U, given that this event would be error-free if the U arose by deamination of C.

In collaboration with Veronica Maher, we set out to test such a hypothesis in vivo. We transfected an XP-V cell line with antisense to POLI and identified two stable cell lines in which pol iota expression was reduced by about 50 percent. As hypothesized, the mutation frequency also decreased by about 50 percent in the antisense-expressing strains. Our results therefore indicate that, in XP-V cells, pol iota does indeed cause the high frequency of mutations and abnormal spectrum induced by UV light.

COLLABORATORS

Jack Ballantyne, PhD, University of Central Florida, Orlando, FL
Jean Cadet, PhD, Laboratoire Lésions des Acides Nucléiques, CEA-Grenoble, Grenoble, France
Pat Gearhart, PhD, Laboratory of Molecular Biology, NIA, Baltimore, MD
Myron F. Goodman, PhD, University of Southern California, Los Angeles, CA
Shigenori Iwai, PhD, Osaka University, Osaka, Japan
Veronica Maher, PhD, Michigan State University, East Lansing, MI
Stephen West, PhD, Cancer Research UK, London, UK

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