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.
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