| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 91 === Nbs1, a DNA double strand break repair protein, forms a complex with Mre11 and Rad50, and so does Xrs2p, the functional homologue of Nbs1 in yeast. There are FHA (forkhead-associated) domain and BRCT (breast cancer carboxy-terminal) domain on the N terminus of Nbs1. Human genetic disease-Nijmegen breakage syndrome (NBS), resulting from NBS1 mutation, has phenotypes such as immunodeficiency, chromosomal instability, radiosensitivity, and high opportunity of cancer producing, similar to the phenotypes of AT (ataxia telangiectasia), ATM-defect disease. When DNA damage occurs in cells, ATM phosphorylates Nbs1, and then cell cycle checkpoint and DNA repair mechanism are activated. Nbs1 can interact with telomeric regulatory proteins, TRF1 and TRF2, and defect on Nbs1 will result in telomere length shortening, which shows that Nbs1 functions on telomere regulation. Besides that the C-terminus of Nbs1 can interact with Mre11, TRF1, TRF2, Lig4, E2F, and the N-terminus of it can interact with γ-H2AX, we found that KPNA2, a member of importin alpha family, interacted with Nbs1 by using yeast two hybrid system, and showed their binding in vivo by immunopercipitation. Doing yeast two hybrid test by truncating Nbs1, a.a. 541~754 deletion on Nbs1 obviously reduced its interacting strength with KPNA2. The a.a. 461 PSTKKRE, which might be NLS of Nbs1, was mutated on Nbs1, and this mutant had nearly no interaction with KPNA2. In addition, we selected Ser397Ala, Lys528 and Gln583Lys these three mutations from sequencing results of reverse yeast two hybrid screen to do site-directed mutagenesis. Testing interaction of generated Nbs1 mutants and KPNA2 by yeast two hybrid, three a.a. -mutated Nbs1 lost its interaction with KPNA2. We also used fluorescence microscopy to observe the distribution of Nbs1 mutants in cells, but all kinds of mutants were localized in the nuclear as wild type Nbs1. In yeast, overexpressing three NbS1 constructs-normal NBS1, NBS1Δ from patient, and NBS1 S343A which can’t be phosphrylated by ATM, we found that normal NBS1 had no effect on the telomeric length of wild type and xrs2 mutant strains, and it looked like that NBS1Δ could slightly lengthen the telomere of xrs2 mutant strain.
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