The broad long-range goals of this project are to understand how two major classes of chemotherapeutic agents cause DNA damage and how cells deal with DNA-protein crosslinks which are induced by these agents. The repair of DNA-protein crosslinks is very poorly understood, in spite of their prevalence from many chemotherapeutic agents as well as radiation and other chemicals. The quinolone antibiotics, which target bacterial DNA gyrase, stabilize a reaction intermediate in which the enzyme is covalently attached to a broken DNA molecule via phosphotyrosine bonds. Anticancer drug 5-azacytidine, on the other hand, leads to covalent complexes between cytosine methyltransferase and DNA at the recognition sites of the enzyme, with no DNA break in the complex. We have shown that both classes of inhibitors lead to blockage of the replication fork at sites where the enzymes are covalently bound to DNA.
The first aim seeks to elucidate the mechanism of DNA breakage and cytotoxicity after treatment with quinolones, with an emphasis on the role of several genes including dnaQ, recQ, xseAB, and ruvAB.
The second aim analyzes mutants that are hypersensitive to 5-azacytidine, which induces covalent methyltransferase-DNA complexes. The basis of their hypersensitivity will be probed with a number of experimental tests, and a major goal is to identify a subset of the mutants affected for repair of DNA damage from the DNA-protein crosslinks.
The third aim uses biochemical approaches to investigate the repair and processing of DNA-protein crosslinks formed by both classes of chemotherapeutic agents. This includes biochemical analyses of intermediates formed in vivo, as well as in vitro experiments where we analyze the fate of DNA-protein crosslinks in reactions with cell extracts or purified proteins. Finally, in the fourth aim, we follow up on our recent observation that the tmRNA translational quality control system is important for survival after 5-azacytidine treatment. We have proposed that the DNA-protein crosslinks block transcription, which leads to blockage of the coupled translating ribosomes, to trigger the tmRNA system into action. Aspects of this model will be tested, including the role of the site- specific crosslinks in triggering the tmRNA system and the fate of the RNA and RNA polymerase in the blocked complexes.

Public Health Relevance

Statement This research is relevant to public health because understanding how chemotherapeutic agents work can help scientists and clinicians improve therapy with the drugs. Two important chemotherapeutic agents are studied here: the quinolones, which are among the most frequently prescribed antibiotics (including the commonly used ciprofloxacin), and 5- azacytidine, which is effective in cancer chemotherapy against leukemia and pre-leukemia syndromes. In addition, the pathways of replication fork failure and the repair of DNA- protein crosslinks are issues relevant to the genome instability that can lead to the formation of cancers.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM072089-08
Application #
8292095
Study Section
Special Emphasis Panel (ZRG1-IDM-S (02))
Program Officer
Reddy, Michael K
Project Start
2004-06-01
Project End
2013-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
8
Fiscal Year
2012
Total Cost
$324,903
Indirect Cost
$116,632
Name
Duke University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
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Kreuzer, Kenneth N (2013) DNA damage responses in prokaryotes: regulating gene expression, modulating growth patterns, and manipulating replication forks. Cold Spring Harb Perspect Biol 5:a012674
Kuo, H Kenny; Krasich, Rachel; Bhagwat, Ashok S et al. (2010) Importance of the tmRNA system for cell survival when transcription is blocked by DNA-protein cross-links. Mol Microbiol 78:686-700
Pohlhaus, Jennifer Reineke; Long, David T; O'Reilly, Erin et al. (2008) The epsilon subunit of DNA polymerase III Is involved in the nalidixic acid-induced SOS response in Escherichia coli. J Bacteriol 190:5239-47
Long, David T; Kreuzer, Kenneth N (2008) Regression supports two mechanisms of fork processing in phage T4. Proc Natl Acad Sci U S A 105:6852-7
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