DNA within eukaryotic cells is continually exposed to DNA damaging agents. These include UV radiation, natural and man-made mutagenic chemicals, mechanical stress on the chromosomes, or by processes such as redox cycling by heavy metal ions and radiomimetic drugs. Of the various forms of damage that are inflicted by these mutagens, probably the most dangerous is the DNA double-strand break (DSB). DSB are generated when the two complementary strands of the double helix are broken simultaneously at sites that are sufficiently close to one another that base pairing and chromatin structure are insufficient to keep the two DNA ends juxtaposed. As a consequence, the ends generated by a DSB are liable to become physically dissociated from one another, making repair difficult and providing an opportunity for inappropriate recombination with other sites in the genome. This inappropriate recombination results in chromosomal instabilities leading to deregulated gene expression and carcinogenesis. To counteract the detrimental effects of these potent lesions, cells have evolved two distinct pathways of DSB repair, homologous recombination (HR) and hon-homologous end joining (NHEJ). NHEJ directly rejoins DSB, whereas HR utilizes a sister chromatid or homologous chromosome as a template for DNA resynthesis and rejoining. Both pathways are highly conserved throughout eukaryotic evolution but their relative importance differs from one organism to another. Simple eukaryotes such as the yeast S.cerevisiae and S.pombe rely mainly on HR to repair radiation-induced DSB. In contrast, in mammals the NHEJ pathway predominates in many stages of the cell cycle, particularly in G0 and G1.
DNA DSB are processed exonucleolytically, yielding 3' overhanging ss-tails of about 600 bases. NHEJ rejoins the two broken ends directly and generally leads to small deletions of DNA sequence. The activity of the Ku70/Ku80 heterodimeric protein is essential to the NHEJ pathway. The Ku heterodimer initiates NHEJ by binding to the free DNA ends and recruiting other NHEJ factors such as DNA-PK, XRCC4 and DNA ligase IV. DNA-PK becomes activated upon DNA binding, and phosphorylates a number of substrates including p53, Ku and the DNA ligase IV cofactor XRCC4. Phosphorylation of these factors further facilitates the repair process. In addition to Ku and DNA ligase IV, the Rad50, MRE11 and NBS1 genes are also involved in NHEJ. Because the ends of most DSB generated by genotoxic agents are damaged and unable to be directly ligated, they often have to undergo limited processing by nucleases and/or polymerases before NHEJ can proceed. The MRE11-Rad50-NBS1 complex, which contains exonuclease, endonuclease and helicase activities, functions in NHEJ particularly if the DNA ends require processing before ligation. Other nucleases are involved in addition to the MRE11 complex, such as Artemis. The final step in NHEJ repair involves ligation of the DNA ends by DNA ligase IV in a complex that also includes XRCC4 and Ku.