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Kenneth N. Kreuzer

Professor Emeritus of Biochemistry
Biochemistry
Duke Box 3711, Durham, NC 27710
155A Nanaline Duke Bldg, Durham, NC 27710

Overview


Our 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 mechanism of phage T4 origin-dependent DNA replication 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.

We are 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 includes important anticancer agents, such as doxorubicin and etoposide, and the fluoroquinolone group 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. Recently, we have extended our studies of anticancer drugs to the nucleotide analog aza-cytosine, which traps covalent methylase-DNA complexes. As with the topoisomerase inhibitors, this drug blocks replication forks at the sites of covalent protein-DNA complexes.

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. The UvsW protein is also important in phage recombination, DNA repair, and mutation. We are collaborating with Dr Stephen White (St. Jude Children's Research Hospital) to analyze the structure and function of the UvsW helicase and to identify its molecular role in recombination and repair.

Current Appointments & Affiliations


Professor Emeritus of Biochemistry · 2015 - Present Biochemistry, Basic Science Departments

Recent Publications


Structural and functional insights into the interaction between the bacteriophage T4 DNA processing proteins gp32 and Dda.

Journal Article Nucleic Acids Res · November 11, 2024 Bacteriophage T4 is a classic model system for studying the mechanisms of DNA processing. A key protein in T4 DNA processing is the gp32 single-stranded DNA-binding protein. gp32 has two key functions: it binds cooperatively to single-stranded DNA (ssDNA) ... Full text Link to item Cite

Functional Analysis of the Bacteriophage T4 Rad50 Homolog (gp46) Coiled-coil Domain.

Journal Article J Biol Chem · September 25, 2015 Rad50 and Mre11 form a complex involved in the detection and processing of DNA double strand breaks. Rad50 contains an anti-parallel coiled-coil with two absolutely conserved cysteine residues at its apex. These cysteine residues serve as a dimerization do ... Full text Link to item Cite
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Recent Grants


Increasing Diversity and Student Engagement in the BioSciences

Institutional SupportCo Investigator · Awarded by Merck Foundation · 2014 - 2015

Processing and consequences of DNA-protein crosslinks in E. coli

ResearchPrincipal Investigator · Awarded by National Institutes of Health · 2002 - 2013

Duke PREP: Minority Recruitment into Biomedical Sciences

Inst. Training Prgm or CMEPrincipal Investigator · Awarded by National Institutes of Health · 2003 - 2008

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Education, Training & Certifications


The University of Chicago · 1978 Ph.D.