Non-homologous End-joining (NHEJ) DNA Repair and the DNA-activated Protein Kinase.
The formation of DNA double-strand breaks (DSBs) is the primary cause of death of mammalian cells in response to ionizing radiation and many anti-cancer agents. DNA DSBs activate cell cycle checkpoints and induce the expression of specific genes; however, the mechanisms by which cells respond to and repair DSBs are incompletely characterized.
A critical enzyme required for the repair of DSBs in vertebrate cells is the DNA-dependent protein kinase, DNA-PK. DNA-PK consists of a very large, 4128 amino acid (~470 kDa), catalytic polypeptide (DNA-PKcs) and a hetero-dimeric DNA-binding subunit, the Ku autoantigen (composed of 70 kDa and 80 kDa polypeptides) that targets DNA-PKcs to DNA ends or structures. DNA-PK belongs to a family of large, phosphoinositide 3-kinase related kinases (PI3KK's) that function in DNA repair and cell cycle regulation, including ATM, the protein defective or missing in patients with the recessive, inherited disease ataxia telangiectasia, ATR, the apparent human homolog of the yeast MEC1p cell cycle checkpoint protein, SMG1 (also called ATX), a kinase involved in nonsense-mediated mRNA decay, and mTOR (also know as FRAP, the FKBP-12-rapamycin-associated protein), a protein required for progression through G1. DNA-PKcs is found mainly in vertebrates (and curiously in the mosquito, honey bee, and sea urchin), whereas other NHEJ components (Ku70, Ku80, XRCC4, DNA Lig4) are present also in plants and lower eukaryotes including C. elegans, Drosophila, yeasts, and distantly related homologues are even present in bacteria.
In vitro DNA-PK is activated by linear double-stranded DNA fragments and DNA structures containing single-to-double strand transitions, but not by closed, fully duplexed, circular DNA. DNA-PK phosphorylates numerous DNA-binding proteins in vitro, including the SV40 large tumor antigen, the single-stranded DNA-binding protein RPA, the tumor suppressor gene product p53 (and many other transcription factors), several DNA repair proteins including XRCC4, the Werner syndrome helicase WRN1, Artemis, and itself. The human DNA-PKcs gene (PRKDC or XRCC7) maps to a locus near the centromere of human chromosome 8. Although defects in DNA-PK have not been associated with a human disease, mice, dogs, and horses with defects in the gene for DNA-PKcs exhibit a severe combined immune deficiency (SCID) phenotype and are sensitive to ionizing radiation. Mice with single amino acid substitutions in DNA-PKcs exhibit increased susceptibility to radiation induced cancer. Drugs that modulate DNA-PK activity or its signal transduction pathway could be important in developing advanced cancer therapies and for modulating immunity.
The p53 Tumor Suppressor Protein
Human p53 is a 393 amino acid, tetrameric transcription factor that is posttranslational modified at approximately 30 different sites by phosphorylation, acetylation, methylation, ubiquitination, neddylation or sumoylation in response to various cellular stress conditions. Genomic approaches indicate that p53 induces or inhibits the expression of 1500 genes. Specific posttranslational modifications are thought to modulate p53 stability, activity as a transcription factor, and promoter selectivity, thus regulating cell fate by controlling the induction of p53-mediated cell cycle arrest, apoptosis, or cellular senescence. Loss of p53 function, either directly through mutation or indirectly through several mechanisms, plays a central role in the development of cancer. Figure 3 summarizes the p53 protein domains, posttranslational modification sites, and the proteins that interact with human p53.
Current efforts are directed at 1) understanding the roles of individual posttranslational modification in regulating p53 activity and stability, and characterizing the genotoxic and non-genotoxic stress pathways that regulate p53 activity, and 2) a whole genome analysis of the chromosomal sites bound by p53 using a newly developed high throughput sequencing approaches..
C.W. Anderson is a founding member and secretary of the
International Association of Protein Structure
Analysis and Proteomics (IAPSAP).