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

Roger Woodgate, PhD, Head, Laboratory of Genomic Integrity
Martha G. Bomar, PhD, Intramural Research Training Award Fellow
Ekaterina Chumakov, PhD, Staff Scientist
Donald Huston, Student Fellow
Wojciech Kuban, PhD, Visiting Fellow
John McDonald, PhD, Biologist
Justyna McIntyre, PhD, Visiting Fellow
Mary McLenigan, BS, Chemist
Catherine Theisen, Student Fellow
Alexandra Vaisman, PhD, Senior Research 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 is 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 synthesis (TLS) or translesion replication (TR), 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.

Mutagenesis in prokaryotes

It has been known for many years that most damage-induced mutagenesis in Escherichia coli depends on the UmuD'2C protein complex, which is DNA polymerase V (polV). Biochemical characterization of polV has been hindered by the fact that the enzyme is notoriously difficult to purify, largely because overproduced UmuC is insoluble. However, over the past 15 years, protocols to purify soluble UmuD'₂C (polV) have improved, but the purification process is nevertheless complex; it requires multiple steps and yields only a few milligrams (at most) of pure protein from hundreds of liters of induced starting culture. A Maltose-Binding Protein (MBP)-UmuC fusion protein has also been purified, but the ~45 kDa MBP-tag is resistant to cleavage, and the recombinant MBP-UmuC fusion protein exhibits other cofactor requirements than does UmuD'₂C. We are interested in further understanding the structural and biochemical basis for polV-dependent translesion DNA synthesis in vitro. To that end, we wish to characterize mutant variants of E. coli polV and its orthologs at the biochemical level. However, we suspected that the existing polV purification protocols would be unlikely to yield sufficient material for our studies. Therefore, we recently developed a novel strategy to purify wild-type polV, mutant polV variants, and polV orthologs rapidly and efficiently in high yield and purity. Indeed, using such an approach, milligram quantities of highly purified polV can be extracted from as little as four liters of starting E. coli culture.

We also developed strategies to purify the E. coli beta-clamp and gamma-clamp loader free of contaminating polymerases. Using these highly purified proteins, we determined the cofactor requirements for optimal activity of efficient and processive DNA synthesis by polV on an approximately 3.0 Kb circular, single-stranded template containing a single cyclobutane pyrimidine dimer (CPD) (see reference 1). We demonstrated that polV shows robust activity on a single-stranded binding (SSB) protein-coated template in the presence of the beta/gamma-complex and a RecA filament in trans. This strong activity was attributed to the unexpectedly high processivity of polVMut (UmuD'₂C·RecA·ATP) (see references 2 and 3), which was efficiently recruited to a primer terminus by SSB.

Development of high-throughput replication assays to identify inhibitors of specialized eukaryotic DNA polymerases

Human cells posses at least 14 DNA polymerases (pols). Three—pol alpha, pol delta, and pol epsilon—are involved in genome duplication. The remaining eleven DNA polymerases have specialized functions within the cell. Four of the specialized DNA polymerases—pol eta, pol iota, pol kappa, and Rev1—belong to the Y-family of DNA polymerases and participate in TLS (see reference 4). Apparently, Pol eta specifically evolved to bypass a CPD, which is the major photoproduct induced upon UV irradiation. Bypass of the CPD is usually efficient and accurate and protects humans from the deleterious consequences of prolonged sunlight exposure. This is evidenced by the fact that humans with defects in POLH, the gene that encodes pol eta, exhibit the variant form of Xeroderma pigmentosum (XP-V). Individuals suffering from XP-V are highly susceptible to sunlight-induced skin damage, with a 2,000-fold greater incidence of skin cancers than in the normal population.

Although pol eta is best characterized for the accuracy and efficiency with which it bypasses CPDs, a number of studies also indicate that it can bypass a variety of other DNA lesions. Of particular relevance is its ability to bypass a cisplatinated intrastrand cross-link. Furthermore, structural studies indicate that the cross-linked adduct can be readily accommodated in the active site of the polymerase. Cisplatin (and related compounds) have been used to successfully treat a number of cancers, such as those of the testis, ovarian, and bladder, for which cancer survival rates are high. However, many tumors often develop drug-resistance, and certain cancers (such as non-small cell lung cancer) do not respond well to cisplatin treatment. Given the ability of pol eta to bypass cisplatinated DNA adducts in vitro, it has been suggested that pol eta–dependent bypass of the cisplatin lesion in vivo increases tumor resistance. Indeed, a direct role in the bypass of cisplatinated DNA lesions appears substantiated by the fact that cells from XP-V patients (devoid of pol eta) are hypersensitive to the killing effects of cisplatin treatment compared with pol eta-proficient cell lines. Furthermore, a recent epidemiological study reported a good correlation between the life expectancy of patients with non-small cell lung cancer and expression levels of pol eta; patients with high levels of pol eta had a life expectancy of 6.9 months after diagnosis, while those with low levels of pol eta lived about three times longer and had a life expectancy 21.1 months after diagnosis.

Thus, while pol eta’s normal function is to protect humans against the deleterious consequences of DNA damage, under certain conditions, it can have deleterious effects on human health. As a consequence, we have collaborated with Anton Simeonov and colleagues (NHGRI) to develop a high-throughput replication assay to identify inhibitors of pol eta (and other DNA polymerase with weak processivity) (see reference 5). We are currently screening over 300,000 compounds with the goal of identifying molecules that specifically inhibit DNA polymerase eta, but not the cell’s replicases (pol alpha, pol delta and pol epsilon). It is hoped that the inhibitory compounds identified during this project will serve as starting points to develop adjuvants to standard chemotherapeutic treatments that will ultimately increase the efficacy of chemotherapy and lead to a better patient prognosis.

Additional Funding

NIH R03 MH090825-01(2010-ongoing) “High Throughput Screening to Identify Inhibitors of Human DNA Polymerase eta”

Publications

Karata K, Vidal AE, Woodgate R. Construction of a circular single-stranded DNA template containing a defined lesion. DNA Repair. 2009;8:852-856.
Jiang Q, Karata K, Woodgate R, Cox MM, Goodman MF. The active form of DNA polymerase V is UmuD'(2)C-RecA-ATP. Nature. 2009;460:359-363.
Patel M, Jiang Q, Woodgate R, Cox MM, Goodman MF. A new model for SOS-induced mutagenesis: how RecA protein activates DNA polymerase V. Crit Rev Biochem Mol Biol. 2010;45:171-184.
Vidal AE, Woodgate R. Insights into the cellular role of enigmatic DNA polymerase iota. DNA Repair. 2009;8:420-423.
Dorjsuren D, Wilson DM 3rd, Beard WA, McDonald JP, Austin CP, Woodgate R, Wilson SH, Simeonov A. A real-time fluorescence method for enzymatic characterization of specialized human DNA polymerases. Nucleic Acids Res. 2009;37:e128.

Collaborators

Myron F. Goodman, PhD, University of Southern California, Los Angeles, CA
Anton Simeonov, PhD, NIH Chemical Genomics Center, NHGRI, Bethesda MD
Antonio E. Vidal, PhD, Instituto de Parasitología y Biomedicina, Granada, Spain
Samuel H. Wilson, MD, Laboratory of Structural Biology, NIEHS, Research Triangle Park, NC

Contact

For more information, email woodgate@mail.nih.gov or visit www.nichd.nih.gov/research/atNICHD/Investigators/woodgate.

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