The repair of Double Strand Breaks (DSB) in DNA is essential for the maintenance of genomic integrity. A growing body of evidence identifies the Non-Homologous End-Joining (NHEJ) mechanism of DSB repair as central to genome maintenance and suggests that deficiencies in the NHEJ apparatus can generate oncogenic events that drive general tumorigenesis. Genetic research has identified many of the key factors that function in mammalian NHEJ. Among these the Ku protein is thought to play an important, but as yet uncharacterized, role. The previous discovery that an inositol phosphate (a small, highly phosphorylated molecule) is bound by Ku and stimulates NHEJ in mammalian cell extracts provides us with a logical starting point from which we plan to investigate the mechanism of this process. In this proposal the roles of Ku and inositol phosphate in the NHEJ reaction will be examined in vitro and in vivo. Despite our understanding of the factors that participate in NHEJ, this process has not yet been reconstituted in vitro. Additionally, a new cell line that is deficient in NHEJ (but normal for all known NHEJ factors) has been discovered. Taken together these data suggests that additional, as yet unidentified, factors may play a role in this process. In addition to our work in characterizing the role of Ku and inositol phosphate we propose to identify additional participants in the mammalian NHEJ reaction. My long-term research aims target the molecular mechanism of DSB repair by NHEJ in mammalian cells. An understanding of this system will provide insight into a mechanism that appears to drive tumor formation and may identify a means of controlling NHEJ in mammalian cells to provide an effective cancer treatment. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM070639-03
Application #
7089000
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Portnoy, Matthew
Project Start
2004-07-01
Project End
2009-03-31
Budget Start
2007-04-01
Budget End
2008-03-31
Support Year
3
Fiscal Year
2007
Total Cost
$254,246
Indirect Cost
Name
Johns Hopkins University
Department
Biochemistry
Type
Schools of Public Health
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Hanakahi, Les (2011) Effect of the inositol polyphosphate InsP(6) on DNA-PK-dependent phosphorylation. Mol Cancer Res 9:1366-76
Cheung, Joyce C Y; Salerno, Brenda; Hanakahi, Les A (2008) Evidence for an inositol hexakisphosphate-dependent role for Ku in mammalian nonhomologous end joining that is independent of its role in the DNA-dependent protein kinase. Nucleic Acids Res 36:5713-26
Jayaram, Sumithra; Ketner, Gary; Adachi, Noritaka et al. (2008) Loss of DNA ligase IV prevents recognition of DNA by double-strand break repair proteins XRCC4 and XLF. Nucleic Acids Res 36:5773-86
Jayaram, Sumithra; Gilson, Timra; Ehrlich, Elana S et al. (2008) E1B 55k-independent dissociation of the DNA ligase IV/XRCC4 complex by E4 34k during adenovirus infection. Virology 382:163-70
Mason, Tracey McGregor; Smeaton, Michael B; Cheung, Joyce C Y et al. (2008) End modification of a linear DNA duplex enhances NER-mediated excision of an internal Pt(II)-lesion. Bioconjug Chem 19:1064-70
Hanakahi, Les A (2007) 2-Step purification of the Ku DNA repair protein expressed in Escherichia coli. Protein Expr Purif 52:139-45
Smeaton, Michael B; Miller, Paul S; Ketner, Gary et al. (2007) Small-scale extracts for the study of nucleotide excision repair and non-homologous end joining. Nucleic Acids Res 35:e152
Baker, Amy; Rohleder, Kent J; Hanakahi, Les A et al. (2007) Adenovirus E4 34k and E1b 55k oncoproteins target host DNA ligase IV for proteasomal degradation. J Virol 81:7034-40