DSB Repair by Non-Homologous End Joining
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DSB Repair by Non-Homologous End Joining

The DNA within our cells is continually being exposed to DNA-damaging agents. These include UV (Ultraviolet Light), natural and man-made mutagenic chemicals, ROS (Reactive Oxygen Species) generated by IR (Ionizing Radiation), replication of ssDNA (single stranded DNA) breaks, mechanical stress on the chromosomes or by processes such as redox cycling by heavy metal ions and radio-mimetic drugs. Of the various forms of damage that are inflicted by these mutagens, probably the most dangerous is the DNA DSB (Double-Strand Break). DNA DSBs are generated when the two complementary strands of the DNA double helix are broken simultaneously at sites that are sufficiently so close to one another that base pairing and chromatin structure are insufficient to keep the two DNA ends juxtaposed. As a consequence, the two DNA ends generated by a DSB are liable to become physically dissociated from one another, making ensuing repair difficult to perform and providing the opportunity for inappropriate recombination with other sites in the genome (Ref.1). 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, HR (Homologous Recombination) and NHEJ (Non-Homologous End Joining). NHEJ directly rejoins DSBs, whereas HR utilizes a sister chromatid or homologous chromosome as a template for DNA resynthesis and rejoining (Ref.2). Both pathways are highly conserved throughout eukaryotic evolution but their relative importance differs from one organism to another. Simple eukaryotes such as the yeasts S.cerevisiae and S.pombe rely mainly on HR to repair radiation-induced DNA DSBs. In contrast, in mammals the NHEJ pathway predominates in many stages of the cell cycle-particularly in G0 and G1 (Ref.1). The cellular decision as to which pathway to utilize for DSB repair is largely influenced by stage within the cell cycle at the time of damage (Ref.3).

DNA DSBs are processed exonucleolytically, yielding 3 overhanging ss-tails of about 600 bases in length. The 3 ssDNA tails formed as a result of break is bound by Rad51 and other recombination factors, which function in concert to locate regions of homology on a corresponding DNA duplex (a homologous chromosome or sister chromatid) and form heteroduplex DNA joints (Ref.4). 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 (DNA-dependent Protein Kinase), XRCC4 to XRCC7 (encoding XRCC4, Ku80, Ku70 and DNA-PKcs proteins, respectively) and DNA Ligase-IV. In addition to the Ku and Ligase-IV homologs, the Rad50, MRE11 (Meiotic Recombination-11) and NBS1 (Nijmegen Breakage Syndrome-1) genes are also involved in NHEJ (Ref.5). 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. Because the ends of most DSBs 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 Rad50-MRE11-NBS1 complex, which contains Exonuclease and Endonuclease and Helicase activities, function in NHEJ, particularly if the DNA ends require processing before ligation. Other nucleases are involved in addition to the MRE11 complex, which include mammalian FEN1 (Flap structure-specific Endonuclease-1), the Artemis protein and S.cerevisiae Rad27p. In many cases, NHEJ may also require the actions of a DNA Polymerase(s) (Ref.1). The final step in NHEJ repair involves ligation of the DNA ends by Ligase-IV in a complex that also includes XRCC4 and Ku.

Unrepaired DSBs can be lethal, whereas misrepaired DSBs can cause chromosomal fragmentation, translocations and deletions. Such lesions are potential inducers of carcinogenesis through activation of proto-oncogenes, inactivation of tumor suppressor genes or loss of heterozygosity. Therefore, effective repair of DSBs is of great importance for the maintenance of genome stability and prevention of carcinogenesis (Ref.6).