Chappell, Claire Joanna; (2002) Double-Strand Break Repair by Non-homologous End-Joining. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Non-homologous end-joining (NHEJ) is an important mechanism of double-strand break repair (DSBR) which is largely conserved from yeast to humans. Mammalian NHEJ is dependent on the Ku, DNA-PKcs, XRCC4 and DNA ligase IV proteins. NHEJ-defective rodent cells are highly sensitive to ionising radiation, implicating NHEJ as a critical mechanism for the repair of radiation-induced double-strand breaks (DSBs). DNA termini at sites of radiation-induced DSBs exhibit modifications, including the presence of 5'-hydroxyl and 3'-phosphate or phosphoglycolate groups. During DSBR, these termini must undergo enzymatic processing to return the normal 5'-phosphate and 3'-hydroxyl groups required for ligation. Despite the activities of the core NHEJ proteins being well documented, little is known about the auxiliary processing factors required for repair of ionising radiation-induced breaks. Using an in vitro DNA end-joining assay catalysed by human cell-free extracts I have investigated the wider protein requirements of NHEJ and the ability of extracts to join certain modified DNA termini. In contrast to the essential role for the Mre11 and Xrs2 proteins in the joining of complementary termini in Saccharomyces cerevisiae, the human homologues, MRE11 and NBS1 were not required for the joining of complementary protruding 5' termini. Joining of modified and non-complementary termini implicated requirements for exonuclease and polymerase activities during extract-catalysed end-joining. Moreover, end-joining reactions with DNA molecules containing 5'-hydroxyl termini demonstrated a role for polynucleotide kinase (PNK) in the repair of radiation-induced breaks. The defective end-joining observed with PNK-depleted extracts was complemented by addition of physiological amounts of purified human PNK, but not T4 PNK. In addition, phosphorylation of 5'-hydroxy termini was not observed when DNA end-joining was blocked, either by use of XRCC4 antibodies or DNA-PKcs-defective extracts, implicating co-ordination between the phosphorylation and end-joining reactions. Finally, gel filtration and coimmunoprecipitation experiments indicated that PNK might be associated with the NHEJ proteins in vivo.

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