The Role of Cysteine Residues of Arabidopsis thaliana Ku Heterodimer in DNA Ends Binding

• Main Authors: Lin, Chueh-Tsun, 林覺圳
• Other Authors: Pan, Rong-Long
• Format: Others
• Language: en_US
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/82528073731666711763

碩士 === 國立清華大學 === 生物資訊與結構生物研究所 === 98 === It is very important for all living organisms to maintain DNA integrity. DNA double-stranded breaks (DSBs) could be caused by a variety of internal or external factors; DSBs are lethal to living organisms. Providing that the loss of genetic information results from failure of the DNA repair, this will lead to the cell death. Hence the repair of DSBs is essential for eukaryotes. Two mainly pathways have evolved to repair and detect the DSBs, homologous recombination (HR) and non-homologous end joining (NHEJ). HR is based on DNA homology, using that as a template to recover genetic information. The procedure for NHEJ is fulfilled by directly ligation of each broken ends, intrinsically making it an error-prone thus. Moreover, NHEJ is preferentially in organisms with large genome. Ku protein was originally identified in Japanese patient serum with autoimmune disease in 1981, and the name Ku was derived from the first two letters of the surname of the prototype. Structurally, Ku protein comprises of two subunits with molecular masses nearly of 70 kDa and 80 kDa respectively and binds noncovalently with each other. In addition to the role in DNA repair, Ku also plays a pivotal role in cellular homeostasis. During NHEJ, Ku is one of the cardinal components to detect and translocate to the ends of termini and Ku is involved in repair of terminal ligation. Additionally, some reports indicated that Ku is in a stable situation and bears physiological activity during the formation of heterodimer. Preceding researches suggested that the double-stranded DNA (dsDNA) end-binding of Ku heterodimer relates to redox situation of cysteine of Ku. In this study, the Arabidopsis thaliana homolog, AtKu70 and AkKu80 were expressed respectively to investigate the modification for residue(s) of cysteine about the binding vicissitude for termini of dsDNA. Electrophoretic mobility shift assay (EMSA) analysis revealed that either of AtKu subunit alone could not bind the double-stranded DNA ends, but an AtKu heterodimer could attain it. AtKu heterodimer was considerably affected by N-ethylmaleimide (NEM) on binding to dsDNA ends when residue(s) of cysteine in AtKu70 was modified solitarily. After formation of AtKu70-AtKu80 complex, addition of NEM further decreased DNA binding affinity. Those phenomena might be involved in a conformational change. To dissect the detailed process, further studies are required.