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Dr. Ken KreuzerProfessor of BiochemistryContact InformationTel. (919) 684-6466 Fax (919) 684-6525 e-mail kenneth.kreuzer@duke.edu Lab LocationRoom 157& 157B Nanaline Duke Mailing AddressBox 3711, DUMC Durham, NC 27710 Education Ph.D. University of Chicago, 1978 Research InterestsOur interests are in the molecular mechanisms of DNA replication, recombination and repair, using bacteriophage T4 and Escherichia coli as model systems. In one project, we are investigating the molecular mechanisms of phage T4 DNA replication, which occurs at early times of infection by an origin-dependent mechanism from RNA-DNA hybrids (R loops). The process of “constitutive stable DNA replication” in E. coli has also been proposed to occur via R-loop intermediates, and we are attempting to isolate and analyze the origins for this mode of DNA synthesis. DNA replication forks often fail during the elongation process, particularly after treatment with agents that cause template DNA damage. One severe form of failure is replication fork breakage, in which one arm of the fork is broken from the branch. Homologous recombination plays a critical role in repairing and restarting these broken replication forks in all cells, and disturbances in this process can lead to genome instability. We have studied double-strand-break directed replication in phage T4, which provides an excellent model system for this process of recombination-dependent DNA replication. Our recent studies on the phage T4 MR (Mre11-Rad50) complex provide evidence that the nuclease activity of the protein is important in this process, and also that the MR complex plays a role in coordinating two broken ends during double-strand break repair. Much interest has recently focused on the physiological roles of DNA helicases, particularly with the discovery of a helicase defect in Bloom’s and Werner’s syndromes in humans, which cause predisposition to cancer. We have shown that the phage T4 UvsW protein is a helicase that unwinds the origin R loops, and provided evidence that this protein is thereby a negative regulator of origin usage at late times of infection. We have also shown that the UvsW protein catalyzes the regression of replication forks, and provided strong evidence that fork regression occurs in vivo. We are collaborating with Dr Stephen White (St. Jude Childrens Research Hospital) to analyze the structure and function of the UvsW helicase and to identify its molecular roles in recombination and repair. We are very interested in the mechanism of cytotoxicity of inhibitors of type II DNA topoisomerases, and we have obtained evidence that cytotoxicity may involve perturbation of replication fork behavior. This group of inhibitors includes important anticancer agents, such as doxorubicin and etoposide, and the fluoroquinolone class of antibacterial agents, such as ciprofloxacin. All of these inhibitors stabilize a reaction intermediate, called the cleavage complex, in which the topoisomerase is covalently attached to cleaved DNA. We have shown that drug-stabilized cleavage complexes block replication forks in vivo both in phage T4 infections, which are sensitive to the anticancer agents, and in uninfected E. coli cells, which are sensitive to the fluoroquinolones. Furthermore, we found that these blocked replication forks are prone to breakage, in at least some cases by recombination nucleases that cut branched DNA. We propose that this “collateral damage” from topoisomerase inhibitors constitutes cytotoxic damage, and may also be involved in genetic rearrangements induced during anticancer chemotherapy. We have extended our studies of anticancer drugs to the nucleotide analog 5-azacytidine, which traps covalent complexes between DNA and cytosine methyltransferase enzymes. As with the topoisomerase inhibitors, we found that this drug blocks replication forks at the sites of the covalent DNA-protein complexes. We are currently studying the detailed mechanism of cytotoxicity from the 5-azacytidine-induced DNA-protein crosslinks using an E. coli model system. Recent Publications1. Long DT, Kreuzer KN. 2009. Fork regression is an active helicase-driven pathway in bacteriophage T4. EMBO Rep. 10:394-399. More… 2. Long DT, Kreuzer KN. 2008. Regression supports two mechanisms of fork processing in phage T4. Proc. Natl. Acad. Sci. USA 105:6852-6857. More… 3. Webb MR, Plank JL, Long DT, Hsieh TS, Kreuzer KN. 2007. The phage T4 protein UvsW drives Holliday junction branch migration. J. Biol. Chem. 282:34401-34411. More… 4. Kuo HK, Griffith JD, Kreuzer KN. 2007. 5-Azacytidine induced methyltransferase-DNA adducts block DNA replication in vivo. Cancer Res. 67:8248-8254. More… 5. Dudas KC, Kreuzer KN. 2005. Bacteriophage T4 helicase loader protein gp59 functions as gatekeeper in origin-dependent replication in vivo. J. Biol. Chem. 280:21561-9 More… 6. Kreuzer KN. 2005. Interplay Between DNA Replication and Recombination in Prokaryotes. Annu. Rev. Microbiol. 59:43-67 More… 7. Pohlhaus JR, Kreuzer KN. 2005. Norfloxacin-induced DNA gyrase cleavage complexes block Escherichia coli replication forks, causing double-stranded breaks in vivo. Mol. Microbiol. 56:1416-29 More… 8. O’Reilly EK, Kreuzer KN. 2004. Isolation of SOS constitutive mutants of Escherichia coli. J. Bacteriol. 186:7149-60 More… 9. Hong G, Kreuzer KN. 2003. Endonuclease cleavage of blocked replication forks: An indirect pathway of DNA damage from antitumor drug-topoisomerase complexes. Proc. Natl. Acad. Sci. U. S. A 100:5046-51 More… 10. Stohr BA, Kreuzer KN. 2002. Coordination of DNA ends during double-strand-break repair in bacteriophage T4. Genetics 162:1019-30 More… |
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