Search

The DNA-dependent protein kinase, DNA-PK, is required for DNA double-strand break repair and V(D)J recombination (1-3). DNA-PK is composed of a large catalytic subunit of approx 460 kDa (DNA-PKcs) and a heterodimeric DNA targeting subunit, Ku. Ku is composed of 70-kDa and approx 80-kDa subunits (called Ku70 and Ku80, respectively) and targets DNA-PKcs to ends of double-stranded DNA. DNA-PKcs is related by amino acid sequence to the phosphatidyl inositol kinase (PI-3 kinase) protein family (4), however, in vitro DNA-PK acts as a serine/threonine protein kinase (5-3). The physiological substrates of DNA-PK are not known, however in vitro DNA-PK is known to phosphorylate many proteins including hsp90, SV40 large T antigen, serum response factor, and p53 (reviewed in ref. 1). In these substrates, DNA-PK phosphorylates serine (or threonine) that is followed by glutamine (an "SQ motif") (8), although some DNA-PK substrates are phosphorylated at "non-SQ" sites (9). A synthetic peptide, derived from amino acids 11-24 of human p53, that contains an "SQ motif" is a specific substrate of DNA-PK, whereas a similar peptide in which the glutamine is displaced from the serine by one amino acid is not phosphorylated (8). These peptides have been used to assay for DNA-PK activity in extracts from human tissue culture cells (8-10) and normal human cells (11). DNA-PK activity is readily detected in extracts from human and monkey cell lines but not in protein extracts from nonprimate cells that, for reasons that are not well understood, contain 50- to 100-fold less DNA-PK activity (8,10,12). DNA-PK activity in nonprimate cells is detected using the same peptide substrates, but using a more sensitive "DNA-cellulose pulldown" assay in which cellular proteins are prebound to double-stranded (ds) DNA cellulose resin and assayed on the resin using the dsDNA cellulose itself as the activator (12,13). Here, we describe these methods for the assay and detection of DNA-PK in extracts from human and rodent cells. It is important to note that both of these assays are useful for quantifying the amount of DNA-PK present that can be activated by exogenous DNA; however, at present, no assay is available to measure the amount of DNA-PK that is actually active in a cell at any particular time. [ABSTRACT FROM AUTHOR]

The electrophoretic mobility shift assay (EMSA) can be used to identify proteins that bind specifically to damaged DNA. EMSAs detect the presence of key DNA repair proteins, such as ultraviolet (UV)-damaged DNA binding protein, which is involved in nucleotide excision repair, and Ku and DNA-PKcs, which are involved in double-strand break repair. This chapter describes EMSA protocols for detecting proteins that bind to UV-damaged DNA, cisplatin-damaged DNA, and DNA ends. The chapter also describes variations of the EMSA that can be used to obtain additional information about these important proteins. The variations include the reverse EMSA, which can detect binding of 35S-labeled protein to damaged DNA, and the antibody supershift assay, which can define the composition of protein-DNA complexes. [ABSTRACT FROM AUTHOR]

Recent genetic and biochemical studies have provided important insights into the mechanism of nonhomologous end-joining (NHEJ) in higher eukaryotes, and have helped to characterize several components including DNA-PKcs, Ku, DNA ligase IV, and XRCC4. There is evidence, however, that additional factors involved in NHEJ remain to be characterized. The biochemical characterization of NHEJ in higher eukaryotes has benefited significantly from in vitro plasmid-based end-joining assays. However, because of differences in the organization and sequence of genomic and plasmid DNA, and because multiple pathways of NHEJ are operational, it is possible that different factors are preferred for the rejoining of double-strand breaks (DSBs) induced in plasmid vs genomic DNA organized in chromatin. Here, we describe an in vitro assay that allows the study of DSB rejoining in genomic DNA. The assay utilizes as a substrate DSBs induced by various means in genomic DNA prepared from agarose-embedded cells after appropriate lysis. Two extremes in terms of state of DNA organization are described: "naked" DNA and DNA organized in chromatin. Here, we describe the protocols developed to carry out and analyze these in vitro reactions, including procedures for preparation of cell extract and the preparation of the substrate DNA ("naked" DNA or nuclei). [ABSTRACT FROM AUTHOR]

Programmed cell death or apoptosis can be induced by a variety of mechanisms including genotoxic stress (1-3). The initiation of apoptosis involves the activation of a proteolytic cascade reminiscent of the blood-clotting pathway or activation of pancreatic proteases (4). It has been suggested that a single DNA strand break or persistent DNA adduct is sufficient to induce apoptosis (5). The protease cascade allows for the amplification of the initial signal and results in the degradation of cellular proteins and chromosomal DNA, which are packaged into apoptotic bodies and subsequently removed and recycled by phagocytic cells. The proteases involved in apoptosis employ active site cysteine residues, which catalyze the hydrolysis of the peptide bond following specific aspartic acid residues (6). This class of proteases has been termed caspases for cysteinyl, aspartate-specific proteases. A current view of the caspase cascade is presented in Fig. 1. Genotoxic stress results in the generation of an as yet undefined signal that results in the release of cytochrome C from the intermembrane space of mitochondria into the cytoplasm. It is in the cytoplasm that cytochrome C can form a complex with apocaspase 9, apoptotic protease activating factor-1 (Apaf-1) and deoxyadenosine 5'triphosphate (dATP). This complex is competent for the autoproteolytic activation of caspase-9 (7). Active caspase-9 then cleaves apocaspase-3 to generate an active caspase-3, which is responsible for cleaving specific target proteins, one of which is the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). The antiapoptotic factor Bcl-xL can sequester cytochrome C and inhibit the formation of the caspase-9-Apaf-1 complex effectively blocking apoptosis (8). The proapoptotic factor Bcl-xS promotes apoptosis by binding to Bcl-xL and thus blocking the inhibitory effect of this protein (8). [ABSTRACT FROM AUTHOR]