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<p> Mre11 and Rad50 form a complex with Nbs1 (MRN) in mammals and Xrs2 (MRX) in yeast. The MRN complex plays a role in many cellular processes, such as DNA damage sensing, DNA repair, cell cycle checkpoint and telomere maintenance. Rad50 contains a conserved ATP binding motif and its ATPase activity is essential for ATM activation in vitro. Using a tethering approach, I have shown that Rad50 can be targeted to telomeres through its fusion to hRap1. The fusion of hRap1 to Rad50 did not alter the property of Rad50. The fused wild-type Rad50 promoted telomerase-dependent telomere lengthening. However, the fusion proteins containing loss-of-function mutations in Rad50 (K42E and S1202R) did not. I have also shown that the fused wild-type Rad50 was able to form irradiation-induced foci in a manner similar to unfused Rad50. In contrast, the two defective mutants of Rad50 failed to accumulate irradiation-induced foci. Expression of the fusion proteins containing Rad50 mutants also interfered with the ability of endogenous Mre11 protein to form foci post irradiation. Thus our data suggest that the Rad50 mutants may function as dominant-negative alleles in cells.</p> / Thesis / Master of Science (MSc)

The polyomavirus enhancer consists of functionally redundant DNA sub-elements. One such sub-element, element 2, comprises a region with contiguous binding sites, or motifs, for at least three nuclear factors, designated as PEA1, PEA2, and PEA3. Although little is known of PEA2, PEA1 is presumed to be a murine homolog of human transcription activator protein 1 (AP-1), and PEA3 has recently been shown to be encoded by a member of the Ets family of oncogenes. The contributions of each factor to enhancer function are not understood. A cell-free system was devised to assay the individual abilities of the DNA motifs recognized by PEA1, PEA2, and PEA3 to confer transcriptional activation upon a minimal promoter. The motifs were cloned and tested as monomers, as multiple tandem copies, and in paired combinations. The results of these in vitro studies indicate that the PEA1 motif behaves as a low affinity AP-1 binding site; that PEA1 and PEA3, but not PEA2, activate transcription; and that both the PEA1 and PEA3 motifs act synergistically. Band shift titration experiments demonstrated that neither PEA1 nor PEA3 bound to their DNA motifs co-operatively, indicating that synergistic activation of transcription by these factors is not due to cooperative binding. Finally, additional in vitro transcription experiments suggest that PEA1 and PEA3 may co-operate with each other to stimulate transcription. A current model proposes that the minimal sub-units of enhancer structure are small (8-10 base pair) DNA motifs, called enhansons, that act synergistically. I propose that the motifs for PEA1 and PEA3, but not PEA2, are enhansons of the polyomavirus enhancer. / Thesis / Master of Science (MS)

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