This application focuses on the pathways of replication fork failure and restart, with a particular emphasis on the response of the fork to site-specific lesions in the template. One type of lesion to be analyzed involves inhibitors of DNA topoisomerases. Several important anticancer drugs, as well as the antibacterial quinolones target type II DNA topoisomerases. These inhibitors stabilize the cleavage complex, a reaction intermediate consisting of the enzyme covalently attached at sites of enzyme-mediated DNA breakage. The cleavage complex is necessary but not sufficient for cytotoxicity, and evidence indicates that DNA replication is important for converting cleavage complexes into cytotoxic and potentially mutagenic lesions. We will attempt to decipher the pathway of cytotoxic lesion generation, in both a phage T4 model system for antitumor drug action and in quinolone-treated Escherichia coli. A second type of lesion involves covalent complexes between cytosine methylases and DNA, but in this case, there is no inherent DNA break. Covalent methylase-DNA complexes are induced at the methylase recognition sites by treatment of cells with aza-cytosine-related compounds, which include antitumor agents active against leukemias and other diseases. ? These compounds have complex mechanisms of action, and the experiments in this application will focus on the consequences of protein-DNA complex formation. We will test the hypothesis that methylase-DNA adducts block the replication fork, and explore the possibility that this fork blockage leads to DNA damage that might be involved in drug cytotoxicity and induced mutagenesis. Finally, abasic sites are a common DNA lesion that occurs under normal growth conditions, and if unrepaired, likely trigger replication fork blockage. We will attempt to deliver DNA with site-specific abasic sites into living E. coli cells, and physically analyze repair and replication fork blockage at these sites. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM072089-02
Application #
6897847
Study Section
Special Emphasis Panel (ZRG1-MBC-2 (01))
Program Officer
Dearolf, Charles R
Project Start
2004-06-01
Project End
2008-05-31
Budget Start
2005-06-01
Budget End
2006-05-31
Support Year
2
Fiscal Year
2005
Total Cost
$277,200
Indirect Cost
Name
Duke University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
Whatley, Zakiya; Kreuzer, Kenneth N (2015) Mutations that Separate the Functions of the Proofreading Subunit of the Escherichia coli Replicase. G3 (Bethesda) 5:1301-11
Krasich, Rachel; Wu, Sunny Yang; Kuo, H Kenny et al. (2015) Functions that protect Escherichia coli from DNA-protein crosslinks. DNA Repair (Amst) 28:48-59
Henderson, Morgan L; Kreuzer, Kenneth N (2015) Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase. PLoS One 10:e0128092
Maduike, Nkabuije Z; Tehranchi, Ashley K; Wang, Jue D et al. (2014) Replication of the Escherichia coli chromosome in RNase HI-deficient cells: multiple initiation regions and fork dynamics. Mol Microbiol 91:39-56
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
Kuo, H Kenny; Griffith, Jack D; Kreuzer, Kenneth N (2007) 5-Azacytidine induced methyltransferase-DNA adducts block DNA replication in vivo. Cancer Res 67:8248-54
Pohlhaus, Jennifer Reineke; Kreuzer, Kenneth N (2006) Formation and processing of stalled replication forks--utility of two-dimensional agarose gels. Methods Enzymol 409:477-93

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