Functional Analysis of Rad50 Mutants

Abstract

<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>