Studies on DNA Replication, Repair, and Mutagenesis in Eukaryotic and Prokaryotic Cells
- Roger Woodgate, PhD, Chief, Section on DNA Replication, Repair, and Mutagenesis
- Ekaterina Chumakov, PhD, Staff Scientist
- Alexandra Vaisman, PhD, Interdisciplinary Scientist
- John McDonald, PhD, Biologist
- Mary McLenigan, BS, Chemist
- Nicholas Ashton, PhD, Postdoctoral Visiting Fellow
- In Soo Jung, PhD, Postdoctoral Visiting Fellow
- Ashley Swanson, PhD, Postdoctoral Intramural Research Training Award Fellow
- Erin Walsh, PhD, Postdoctoral Intramural Research Training Award Fellow
- Donald Huston, BS, Technical Intramural Research Training Award Trainee
- Corinne Farrell, BS, Postbaccalaureate Intramural Research Training Award Fellow
- Kristiana Moreno, Stay-in-School Intramural Research Training Award 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, 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, which 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.
Eukaryotic DNA repair and mutagenesis
The cDNA encoding human DNA polymerase iota (POLI) was cloned in 1999. At that time, it was believed that the POLI gene encoded a protein of 715 amino acids. Advances in DNA sequencing technologies lead to the realization that there is an upstream, in-frame initiation codon that would encode a DNA polymerase iota (pol iota) protein of 740 amino acids. The extra 25 amino–acid region is rich in acidic residues (11/25) and is reasonably conserved in eukaryotes ranging from fish to humans. As a consequence, the curated Reference Sequence (RefSeq) database identified pol iota as a 740 amino–acid protein. However, the existence of the 740 amino–acid pol iota has never been shown experimentally. Using highly specific antibodies to the 25 N-terminal amino acids of pol iota, we were unable to detect the longer 740 amino–acid (iota-long) isoform in western blots. However, trace amounts of the iota-long isoform were detected after enrichment by immunoprecipitation. One might argue that the longer isoform has a distinct biological function, if it exhibits significant differences in its enzymatic properties from the shorter, well characterized 715 amino–acid pol iota. We therefore purified and characterized recombinant full-length (740 amino acid) pol iota-long and compared it with full-length (715 amino–acid) pol iota-short in vitro. The metal ion requirements for optimal catalytic activity differ slightly between iota-long and iota-short, but under optimal conditions, both isoforms exhibit indistinguishable enzymatic properties in vitro. We also reported that, like iota-short, the iota-long isoform can be mono-ubiquitinated and poly-ubiquitinated in vivo, as well as form damage-induced foci in vivo. We conclude that the predominant isoform of DNA pol iota in human cells is the shorter 715 amino–acid protein and that if, or when, expressed the longer 740 amino–acid isoform has identical properties to the considerably more abundant shorter isoform.
In 2003, we reported that mice derived from the 129 strain (129-derived strain) carry a naturally occurring nonsense mutation at codon 27 of the Poli gene that would produce a pol iota peptide of just 26 amino acids, rather than the full-length 717 amino–acid wild-type polymerase. In support of the genomic analysis, no pol iota protein was detected in testes extracts from 129X1/SvJ mice, where wild-type pol iota is normally highly expressed. The early truncation in pol iota occurs before any structural domains of the polymerase are synthesized and, as a consequence, we reasoned that 129-derived strains of mice should be considered as functionally defective in pol iota activity. However, it was recently reported that, during the maturation of the Poli mRNA in 129-derived strains, exon-2 is sometimes skipped and that an exon-2–less pol iota protein of 675 amino acids is synthesized that retains catalytic activity in vitro and in vivo. From a structural perspective, we found this idea untenable, given that the amino acids encoded by exon-2 include residues critical for the coordination of the metal ions required for catalysis, as well as the structural integrity of the DNA polymerase. To determine whether the exon-2–less pol iota isoform possesses catalytic activity in vitro, we purified a glutathione-tagged full-length exon-2–less (675 amino–acid) pol iota protein from baculovirus-infected insect cells and compared the activity of the isoform to full-length (717 amino–acid) GST–tagged wild-type mouse pol iota in vitro. Reactions were performed under a range of magnesium or manganese concentrations, as well as various template sequence contexts. Wild-type mouse pol iota exhibited robust characteristic properties previously associated with human pol iota’s biochemical properties. However, we did not detect any polymerase activity associated with the exon-2–less pol iota enzyme under the same reaction conditions and concluded that exon-2–less pol iota is indeed rendered catalytically inactive in vitro.
Additional Funding
- Collaborative extramural U01 grant (U01HD085531-01) with Prof. Digby Warner, University of Cape Town, South Africa: Replisome dynamics in M. tuberculosis: linking persistence to genetic resistance
Publications
- Frank EG, McDonald JP, Yang W, Woodgate R. Mouse DNA polymerase iota lacking the forty-two amino acids encoded by exon-2 is catalytically inactive in vitro. DNA Repair 2017 50:71-76.
- Frank EG, McLenigan MP, McDonald JP, Huston D, Mead S, Woodgate R. DNA polymerase iota: the long and the short of it! DNA Repair 2017 58:47-51.
- Lee D, An J, Park YU, Liaw H, Woodgate R, Park JH, Myung K. SHPRH regulates rRNA transcription by recognizing the histone code in an mTOR-dependent manner. Proc Natl Acad Sci USA 2017 114:E3424-3433.
- Vaisman A, Woodgate R. Translesion DNA polymerases in eukaryotes: what makes them tick? Crit Rev Biochem Mol Biol 2017 52:274-303.
Collaborators
- Michael Cox, PhD, University of Wisconsin, Madison, WI
- Patricia J. Gearhart, PhD, Laboratory of Molecular Biology and Immunology, NIA, Baltimore, MD
- Myron F. Goodman, PhD, University of Southern California, Los Angeles, CA
- Kyungjae Myung, PhD, Ulsan National Institute of Science and Technology, Ulsan, South Korea
- Andrew Robinson, PhD, University of Wollongong, Wollongong, Australia
- Anton Simeonov, PhD, Scientific Director, NCATS, Bethesda, MD
- Antoine Van Oijen, PhD, University of Wollongong, Wollongong, Australia
- Digby Warner, PhD, University of Cape Town, Cape Town, South Africa
- Wei Yang, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
Contact
For more information, email woodgate@.nih.gov or visit sdrrm.nichd.nih.gov.
