This proposal will examine the biochemical function of the protein complex made up of hMre 11, hRad5O and p95 (MR95). Although much is known concerning the basic activities of the complex, the ability of the complex to interact with chromatin substrates, the mechanistic basis of the DNA binding activity and the contribution of various domains of Nbs 1 are not understood. In order to shed light on these questions, we propose the following specific aims.
Specific Aim 1 : To examine the effect of nucleosomal DNA on the exonuclease activity of the MR95 complex. This will be carried out by using chromatinized substrate double-stranded DNA with the full MR95 complex in exonuclease assays.
Specific Aim 2 : To investigate the ability of the MR95 complex to simultaneously bind to two molecules of DNA. This will be carried out using a pull down assay with one DNA substrate biotinylated. Following the addition of MRN and a second substrate molecule, we will pull down this complex with streptavidin beads. The presence of the non-biotinylated substrate will be assessed by PCR.
Specific Aim 3 : To examine the contribution of p95 to biochemical activities of MR95. Recently a truncated form of Nbsl has been shown to partially complement the phenotypic defects of NBS. In order to fully understand these in vivo results, we will examine the contribution of full-length as well as various truncation mutations of p95 to nuclease activity of the MR95 complex. The execution of these specific aims will enhance the level of understanding of the mechanistic action of the MRN complex and provide insight into its ability to function in multiple DNA metabolic pathways.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA087851-02
Application #
6621760
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Pelroy, Richard
Project Start
2002-01-10
Project End
2005-12-31
Budget Start
2003-01-01
Budget End
2003-12-31
Support Year
2
Fiscal Year
2003
Total Cost
$246,881
Indirect Cost
Name
University of Maryland Baltimore
Department
Radiation-Diagnostic/Oncology
Type
Schools of Medicine
DUNS #
188435911
City
Baltimore
State
MD
Country
United States
Zip Code
21201
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