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National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2020 Annual Report of the Division of Intramural Research

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

Roger Woodgate
  • Roger Woodgate, PhD, Chief, Section on DNA Replication, Repair, and Mutagenesis
  • Alexandra Vaisman, PhD, Interdisciplinary Scientist
  • John P. McDonald, PhD, Biologist
  • Mary McLenigan, BS, Chemist
  • Nicholas Ashton, PhD, Postdoctoral Visiting Fellow
  • Mallory R. Smith, PhD, Postdoctoral Intramural Research Training Award Fellow
  • Katherine Mnuskin, BS, Postbaccalaureate Intramural Research Training Award Fellow
  • Dominic R. Quiros, BS, Postbaccalaureate Intramural Research Training Award Fellow
  • Nicole Wilkinson, BS, Postbaccalaureate Intramural Research Training Award Fellow
  • Maya Kaplan, 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 trans-lesion 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 (pol IV/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.

Prokaryotic studies

As part of an international scientific collaboration with Andrew Robinson, Myron Goodman, and Michael Cox, we investigated the role of Escherichia coli DNA polymerase IV (pol IV) in double strand break repair. To do so, Andrew Robinson’s group used live-cell single-molecule microscopy with fluorescently tagged pol IV and found that exposure to ciprofloxacin and trimethoprim antibiotics leads to the formation of double strand breaks in E. coli cells that strongly stimulate pol IV activity. Furthermore, the RecA recombinase and pol IV foci increase after antibiotic treatment and exhibit strong colocalization. Interestingly, the induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci, and RecA-pol IV colocalization, all depend on RecB function. We hypothesized that the positioning of pol IV foci likely reflects a physical interaction with nucleoprotein filaments denoted RecA* that was detected previously in vitro. Our observations therefore provided an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break–inducing antibiotics [Reference 1].

Eukaryotic studies

Maintaining the genomic integrity of cells is vital, as alterations to the genetic code can result in deregulation of cellular function, malignant transformation, or cell death. This can lead to a variety of disorders including neurological degeneration, premature aging, developmental defects and cancer. To prevent genetic alterations, cells employ a range of genome-stability pathways, which allow for the accurate metabolism of the DNA, as well as for any DNA errors or damage to be rapidly repaired. Post-translational modifications play an essential role in the signaling, activation and coordination of the genome stability pathways [References 2 & 3]. The reversible ubiquitination of proteins is one such essential modification. Ubiquitination is mediated by a cascade of E1, E2, and E3 ubiquitin enzymes, which covalently attach the 8.5 kDa ubiquitin protein onto a substrate molecule, while deubiquitinating enzymes (DUBs) can edit or remove ubiquitin modifications.

Human DNA polymerase iota (pol iota) was discovered by scientists in our lab two decades ago, yet its cellular function remains enigmatic [Reference 4]. As part of our ongoing research on pol iota, we previously reported that the enzyme is ubiquitinated at over 27 individual sites in the 715 amino acid protein. In collaborative studies with Irina Bezsonova, we have now identified Ubiquitin-Specific Protease 7 (USP7) as the enzyme that de-ubiquitinates pol iota. This is extremely interesting, as USP7 has recently emerged as a key regulator of ubiquitination in the genome stability pathways because of its extensive network of interacting partners and established roles in cell-cycle activation, immune responses, and DNA replication. USP7 is also deregulated in many cancer types, where deviances in USP7 protein levels are correlated with cancer progression.

USP7 contains of an N-terminal tumor necrosis receptor-associated factor (TRAF)–like domain, a catalytic domain, and five C-terminal ubiquitin-like domains (UBLs). While the catalytic domain mediates the enzymatic function of the protein, the TRAF-like and UBL domains are essential for substrate specificity and enzymatic activation. These functions are mediated by protein-binding sites, located on TRAF and the first and second UBL domain (UBL1–2). Interestingly, while all other characterized USP7 substrates bind to one or the other protein-binding sites, our studies with DNA polymerase iota revealed that a novel USP7 substrate interacts with both domains. Using biophysical approaches and mutational analysis, we characterized both interfaces and demonstrated that bipartite binding to both USP7 domains is required for efficient DNA polymerase iota de-ubiquitination. Taken together, our data established a new bipartite mode of USP7–substrate binding [Reference 5].


  1. Henrikus SS, Henry C, McGrath AE, Jergic S, McDonald JP, Hellmich Y, Bruckbauer ST, Ritger ML, Cherry ME, Wood EA, Pham PT, Goodman MF, Woodgate R, Cox MM, van Oijen AM, Ghodke H, Robinson A. Single-molecule live-cell imaging reveals RecB-dependent function of DNA polymerase IV in double strand break repair. Nucleic Acids Res 2020;48:8490-8508.
  2. Valles GJ, Bezsonova I, Woodgate R, Ashton NW. USP7 is a master regulator of genome stability. Front Cell Dev Biol 2020;8:717.
  3. Wilkinson NA, Mnuskin KS, Ashton NW, Woodgate R. Ubiquitin and Ubiquitin-like proteins are essential regulators of DNA damage bypass. Cancers (Basel) 2020;12:2848.
  4. Vaisman A, Woodgate R. DNA polymerase iota remains enigmatic 20 years after its discovery. DNA Repair (Amst) 2020;93:102914.
  5. Ashton NW, Valles GJ, Jaiswal N, Bezsonova I, Woodgate R. DNA polymerase iota interacts with both the TRAF-like and UBL1-2 domains of USP7. J Mol Biol 2021;433(2):166733.


  • Irina Bezsonova, PhD, University of Connecticut, Farmington, CT
  • Anders R. Clausen, PhD, Göteborgs Universitet, Göteborg, Sweden
  • Michael Cox, PhD, University of Wisconsin, Madison, WI
  • Iwona Fijlakowska, PhD, Polish Academy of Sciences, Warsaw, Poland
  • Myron F. Goodman, PhD, University of Southern California, Los Angeles, CA
  • Karolina Makiela-Dzbenska, PhD, Polish Academy of Sciences, Warsaw, Poland
  • Justyna McIntyre, PhD, Polish Academy of Sciences, Warsaw, Poland
  • Andrew Robinson, PhD, University of Wollongong, Wollongong, Australia
  • Ewa Sledziewska-Gojska, PhD, Polish Academy of Sciences, Warsaw, Poland
  • 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


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