Browsing by Author "Radhakrishnan, Sarvan Kumar"

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    A Tale of a ‘Tail’ – Understanding the Role of Ku80 C-terminal Region in Non-Homologous End Joining
    (2015-12-14) Radhakrishnan, Sarvan Kumar; Lees-Miller, Susan; Cobb, Jennifer; MacDonald, Justin
    Non-homologous end joining (NHEJ) is the major DNA double strand (DSB) break repair pathway in mammalian cells. The first step in NHEJ is recognition of DSBs by the Ku heterodimer and subsequent recruitment of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a serine/threonine protein kinase, to form the DNA-PK complex. This complex aligns DSB ends, activates Artemis endonuclease activity and finally recruits XRCC4-DNA ligase IV, which ligates DNA ends. The Ku heterodimer consists of 70 and 80 kDa subunits and is conserved throughout evolution. It has been suggested that the extreme C-terminal 14 amino acids of Ku80 is required for DNA-PKcs recruitment and activation. However, another study demonstrated that deletion of the Ku80 C-terminal region (CTR) does not abolish DNA-PKcs activation. Thus, there is considerable ambiguity regarding the role of the Ku80 CTR in DNA-PKcs recruitment and activation. The aim of this study is to understand the role of Ku80 CTR in NHEJ with focus on its ability to recruit and activate DNA-PKcs kinase activity. Using clonogenic cell survival assays, I confirmed that hamster cells expressing Ku80 CTR deletions are radiosensitive and also showed sensitivity to other DSB inducing agents such as doxorubicin and neocarzinostatin. I then generated Ku80 C-terminal deletions (Ku80 residues 1-718 and 1-569), cloned them into baculovirus vectors and expressed and purified the corresponding Ku heterodimers from insect cells. In vitro autophosphorylation reactions, in presence of calf-thymus DNA, using purified proteins showed that Ku heterodimer with Ku80 residues 1-718 showed only a slight defect in DNA-PKcs autophosphorylation, whereas heterodimer with Ku80 residues 1-569 had significant defects in multiple DNA-PKcs autophosphorylation sites. Surprising results were observed when defined DNA structures such as 25 base pair (bp) blunt ended double stranded (ds) DNA was used. Deletion of the entire Ku80 CTR (residues 570-732) lead to abrogation of DNA-PKcs kinase activity and inability to interact with DNA-PKcs protein. On the other hand, deletion of extreme C-terminal 14 amino acids of Ku80 did not affect DNA-PKcs kinase activity but showed defects in its ability to interact with DNA-PKcs. These defects may underlie the radiation and chemosensitivity of Ku80 CTR deletion mutants.