Exposure to a variety of chemical agents that arise from both endogenous and exogenous sources, as well as exposure to ionizing and ultraviolet radiation, produces crosslinks between DNA and protein molecules, in addition to the more extensively characterized individual DNA or protein adducts. All of these agents are either known or suspected carcinogens and as such, pose significant health risks to exposed human populations. Despite the pervasiveness of these DNA-protein crosslinks (DPCs) and their probable causative role in some cancers, age-related macular degeneration and some neurodegenerative diseases such as Parkinson's, there exists a paucity of data concerning the biological processing of these lesions, because until recently, there have been no mechanisms available to create site-specific, protein-specific lesions in DNA. In order to establish a fundamental understanding of cellular responses to the presence of DPCs, herein for the first time, methodologies are developed that create site-specific DPCs that are located in the major and minor grooves of DNA and along the sugar-phosphate backbone. The availability of DNAs containing site-specific and protein-specific DPCs will enable the testing of hypotheses on how these lesions are replicated and repaired in mammalian cells.
Specific Aim (1) lays the foundation for the construction and physical characterization of defined DNAs containing a variety of DPC lesions; strategies are developed to create various sized DPCs (600 - 120,000 Da) in DNAs and characterize their modulation of the DNA structure by analyses of bend angles, footprint, and thermal destabilization.
Specific Aim (2) will characterize in vitro replication and repair of DNAs containing these DPC lesions, using both prokaryotic and mammalian NER assays, DNA replication bypass analyses, and DNA helicase unwinding studies.
Specific Aim (3) evaluates the mutagenic potential and in vivo repair pathways for DPCs by engineering these lesions into single- and double-stranded vectors and replicating them through repair proficient and deficient cells; mutational spectra will be analyzed for these site-specific lesions.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA106858-01
Application #
6763592
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Okano, Paul
Project Start
2004-06-01
Project End
2008-05-31
Budget Start
2004-06-01
Budget End
2005-05-31
Support Year
1
Fiscal Year
2004
Total Cost
$278,595
Indirect Cost
Name
Oregon Health and Science University
Department
Neurosciences
Type
Schools of Medicine
DUNS #
096997515
City
Portland
State
OR
Country
United States
Zip Code
97239
Kumari, Anuradha; Owen, Nichole; Juarez, Eleonora et al. (2015) BLM protein mitigates formaldehyde-induced genomic instability. DNA Repair (Amst) 28:73-82
Kasiviswanathan, Rajesh; Minko, Irina G; Lloyd, R Stephen et al. (2013) Translesion synthesis past acrolein-derived DNA adducts by human mitochondrial DNA polymerase ?. J Biol Chem 288:14247-55
Yamanaka, Kinrin; Dorjsuren, Dorjbal; Eoff, Robert L et al. (2012) A comprehensive strategy to discover inhibitors of the translesion synthesis DNA polymerase ?. PLoS One 7:e45032
Kumari, Anuradha; Lim, Yun Xin; Newell, Amy Hanlon et al. (2012) Formaldehyde-induced genome instability is suppressed by an XPF-dependent pathway. DNA Repair (Amst) 11:236-46
Huang, Hai; Wang, Hao; Voehler, Markus W et al. (2011) ?-Hydroxy-1,N2-propano-2'-deoxyguanosine DNA adduct conjugates the N-terminal amine of the KWKK peptide via a carbinolamine linkage. Chem Res Toxicol 24:1123-33
Yamanaka, Kinrin; Minko, Irina G; Finkel, Steven E et al. (2011) Role of high-fidelity Escherichia coli DNA polymerase I in replication bypass of a deoxyadenosine DNA-peptide cross-link. J Bacteriol 193:3815-21
Kumari, Anuradha; Minko, Irina G; Smith, Rebecca L et al. (2010) Modulation of UvrD helicase activity by covalent DNA-protein cross-links. J Biol Chem 285:21313-22
Huang, Hai; Kozekov, Ivan D; Kozekova, Albena et al. (2010) Minor groove orientation of the KWKK peptide tethered via the N-terminal amine to the acrolein-derived 1,N2-gamma-hydroxypropanodeoxyguanosine lesion with a trimethylene linkage. Biochemistry 49:6155-64
Yamanaka, Kinrin; Minko, Irina G; Takata, Kei-ichi et al. (2010) Novel enzymatic function of DNA polymerase nu in translesion DNA synthesis past major groove DNA-peptide and DNA-DNA cross-links. Chem Res Toxicol 23:689-95
de Graaf, Bendert; Clore, Adam; McCullough, Amanda K (2009) Cellular pathways for DNA repair and damage tolerance of formaldehyde-induced DNA-protein crosslinks. DNA Repair (Amst) 8:1207-14

Showing the most recent 10 out of 14 publications