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
- Erin Walsh, PhD, Postdoctoral Intramural Research Training Award Fellow
- 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.
Prokaryotic mutagenesis
As part of an international scientific collaboration with Andrew Robinson, Antoine van Oijen, Myron Goodman, and Michael Cox, we investigated the sub-cellular localization of the TLS polymerase pol IV in Escherichia coli. DNA polymerase pol IV is one of three TLS polymerases found in E. coli. All three polymerases are produced at elevated levels in bacteria as part of the SOS response to DNA damage. Historically, they have been thought to serve as a last-resort DNA damage–tolerance mechanism, re-starting replication forks that have stalled at damage sites on the DNA. TLS polymerases are highly error prone: inducing their activities leads to increased rates of mutation (error rates of up to 1 in every 100 nucleotides incorporated into DNA). TLS is an important source of mutations that fuel bacterial evolution. For several species of bacteria, deleting genes for TLS polymerases dramatically reduces rates of antibiotic resistance development in laboratory measurements, and in some cases even reduces infectivity. Many of the drugs used to treat bacterial infections cause an increase in mutation rates as a result of TLS. It remains unclear, however, whether TLS polymerases contribute to resistance by providing damage tolerance, increasing cell survival, and thus the chances that a resistant mutant will be found, or by facilitating adaptive mutation, i.e., selectively increasing mutation rates to speed the evolution of drug resistance.
It is thought that pol IV is the most abundant TLS polymerase in E. coli. From Western blots, it has been estimated that levels of pol IV increase from approximately 250 molecules per cell in the absence of DNA damage to 2,500 molecules per cell upon activation of the SOS damage response. On a variety of different lesion-containing DNA substrates pol IV promotes TLS, although its tendency for misincorporation varies with lesion type. The polymerase bypasses adducts to the N2 position of guanines and a variety of alkylation lesions in a mostly error-free fashion. When overexpressed, pol IV induces -1 frameshift mutations in cells treated with alkylating agents. In addition to these lesion bypass activities, pol IV participates in transcription and double-strand break repair and contributes significantly to cell fitness in late stationary phase cultures in the absence of any exogenous DNA damage. It is also been reported that pol IV is required for the formation of adaptive point mutations in the lac operon and was found to be a major determinant in the development of ciprofloxacin resistance in a laboratory culture model.
Visualization of pol IV within live bacterial cells would make it possible to better understand how pol IV activity is regulated in response to DNA damage and test proposed models for its TLS activity at replisomes. To do so, we reported a single-molecule time-lapse approach to investigate pol IV dynamics and kinetics in live E. coli cells under normal growth conditions and following treatment with the antibiotic ciprofloxacin, the DNA–damaging agent MMS, or ultraviolet (UV) light. Twenty minutes after treating cells with the DNA–damaging antibiotic ciprofloxacin, we observed a striking increase in pol IV fluorescence, indicative of SOS–dependent up-regulation. During the same period, we observed an increase in the formation of punctate foci, consistent with individual molecules of pol IV binding to DNA. The canonical view in the field is that pol IV primarily acts at replisomes that have stalled on the damaged DNA template. In contrast with this view, we observed that only a small proportion (about 10%) of pol IV foci colocalize with replisome markers. Initially, the proportion of replisomes that contain pol IV tracked with the increasing concentration of pol IV. In the period 90–180 min after ciprofloxacin addition, however, colocalization dropped dramatically, despite pol IV concentrations remaining relatively constant. Our data strongly suggest that pol IV is only licensed to carry out TLS at stalled replication forks during the early stages of SOS, whereas it continues to act on substrates outside of replication forks throughout the SOS response. In an SOS–constitutive mutant that expressed high levels of pol IV, we observed few foci in the absence of damage, indicating that access of pol IV to DNA is dependent on the presence of damage, as opposed to concentration-driven competition for binding sites.
As part of a scientific collaboration with Martin Gonzalez, we investigated how the mutagenesis-promoting activity of the E. coli polV ortholog polVICE391 is normally kept to a minimum. polVICE391 demonstrates a higher frequency of spontaneous mutagenesis and transversion mutations than the other cloned polV orthologs that have been previously characterized. However, the high mutation frequency promoted by polVICE391 is only evident with the sub-cloned operon and is not seen when it is expressed from the native 88kb Integrating Conjugative Element ICE391. ICE391 is a mobile genetic element capable of transfer between different species of bacteria. Therefore, it seemed plausible that ICE391 carries factor(s) necessary to minimize aberrant polVICE391–mediated mutagenesis. In particular, we identified SetRICE391 as a regulator of the polVICE391–encoded rumAB operon, given that E.coli expressing SetRICE391 demonstrated lower levels of polVICE391–mediated spontaneous mutagenesis than cells lacking SetRICE391. Electrophoretic mobility shift assays revealed that SetRICE391 acts as a transcriptional repressor of polVICE391 by binding to a site overlapping the –35 region of the rumAB operon promoter. SetRICE391 regulation was shown to be specific for the rumAB operon, and in vitro studies with highly purified SetRICE391 revealed that, under alkaline conditions, as well as in the presence of activated RecA, SetRICE391 undergoes a self-mediated cleavage reaction that inactivates its repressor functions. Conversely, a non-cleavable SetRICE391 mutant capable of maintaining repressor activity, even in the presence of activated RecA, exhibited low levels of polVICE391–dependent mutagenesis. Our studies therefore provided compelling evidence that SetRICE391 acts as a transcriptional repressor of the ICE391–encoded mutagenic response in vivo.
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
- Henrikus SS, Wood EA, McDonald JP, Cox MM, Woodgate R, Goodman MF, van Oijen AM, Robinson A. DNA polymerase IV primarily operates outside of DNA replication forks in Escherichia coli. PloS Genetics 2018;14:e018454.
- Vaisman A, Woodgate R. Ribonucleotide discrimination by translesion synthesis DNA polymerases. Crit Rev Biochem Mol Biol 2018;53:382-402.
- Gonzalez M, Huston D, McLenigan MP, McDonald JP, Garcia AM, Borden KS, Woodgate R. SetR-ICE391, a negative transcriptional regulator of the Integrating Conjugative Element 391 mutagenic response. DNA Repair 2018;73:99-109.
Collaborators
- Michael Cox, PhD, University of Wisconsin, Madison, WI
- Martin Gonzalez, PhD, Southwestern University, Georgetown, TX
- Myron F. Goodman, PhD, University of Southern California, Los Angeles, CA
- 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.
