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The DNA-dependent protein kinase plays a critical role in mammalian DNA double strand break (DSB) repair and in specialized recombination, such as lymphoid V(D)J recombination. Its regulatory subunit Ku (dimer of the Ku70 and Ku80 protein) binds to DNA and recruits the kinase catalytic sub-unit, DNA-PKcs. We show here that three different strains deficient in either the Ku80 (xrs-6) or DNA-PKcs (V-3, scid) component of DNA-PK are markedly sensitive (3.5- to 5-fold) to a group of DNA cross-linking agents, the nitrogen mustards (NMs) (melphalan and mechlorethamine) as compared to their parental cell line. Importantly, the level of hypersensitivity to these drugs was close to the level of hypersensitivity observed for radiomimetic agents that create DSBs in DNA (bleomycin and neocarzinostatin). In addition, sensitivity to NMs was restored to the parental level in the xrs-6 cell line stably transfected with the human Ku80 gene (xrs-6/Ku80), showing unequivocally that DNA-PK is involved in this phenotype. These results indicate that a function of the whole DNA-PK protein complex is involved in the cellular response to NMs and suggest that the repair of DNA interstrand cross-links induced in DNA by NMs involved a DNA-PK dependent pathway that shares common features with DNA DSBs repair.

The DNA-dependent protein kinase (DNA-PK) is a trimeric nuclear serine/threonine protein kinase consisting of a large catalytic sub-unit and the Ku heterodimer that regulates kinase activity by its association with DNA. DNA-PK is a major component of the DNA double strand break repair apparatus, and cells deficient in one of its component are hypersensitive to ionizing radiation. DNA-PK is also required to lymphoid V(D)J recombination and its absence confers in mice a severe combined immunodeficiency phenotype. The purpose of this review is to summarize the current knowledge on the mechanisms that contribute to regulate DNA-PK activity in vivo or in vitro and relates them to the role of DNA-PK in cellular functions. Finally, the studies devoted to drug-inhibition of DNA-PK in order to enhance cancer therapy by DNA-damaging agents are presented.

The development of in vitro repair assays with human cell-free extracts led to new insights on the mechanism of excision of DNA damage which consists of incision/excision and repair synthesis/ligation. We have adapted the repair synthesis reaction with cells extracts incubated with damaged plasmid DNA performed in liquid phase to solid phase by DNA adsorption into microplate wells. Since cells extracts are repair competent in base excision and nucleotide excision repair, all types of substrate DNA lesions were detected with chemiluminescence measurement after incorporation of biotin-deoxynucleotide during the repair synthesis step. Derivatives of our initial 3D-assay (DNA damage detection) have been set up to: i) screen antioxidative compounds and NER inhibitors; ii) capture genomic DNA (3D(Cell)-assay) that allows detection of alkylated base and consequently determines the kinetics of the cellular repair; and iii) immunodetect the repair proteins in an ELISA reaction (3D(Rec)-assay). The 3D derived assays are presented and discussed.

Great progress is being made in understanding the process of nucleotide excision repair (NER) in eukaryotes. Different lines of research have been developed, among them an in vitro assay with cell-free extracts has played a major role. This in vitro repair assay takes advantage of a cell-free system that can mediate DNA excision-repair by transcriptionally active protein extracts from mammalian cells incubated in the presence of two plasmids of different sizes, one damaged and the other undamaged as internal control. The extent of repair activity is determined by following the level of radiolabeled incorporation during the repair synthesis step consecutive to the excision of DNA lesions. We discuss the interest and drawbacks of this biochemical assay in light of the main results obtained. We report the modifications that we have undertaken in order to determine repair synthesis activity in a chemiluminescent-directed reaction as well as to assess incision activity in protein extracts.

Using a two-site immunometric assay (IRMA) we quantified the concentration of single-stranded DNA binding protein (SSB) in several E. coli strains. We found approximately 7,000 monomers of SSB present per bacterium, and this number remained constant throughout the exponential phase of growth. Two ssb- mutants (ssb-1 and ssb-113) are defective in the induction of the S.O.S. pathway. One of the first functions expressed upon induction of the S.O.S. pathway is the amplification of recA protein (RECA), which we monitored by an IRMA assay similar to the one used for SSB quantification. By combining the two assays we determined the level of SSB and RECA in ssb- mutants or in SSB and RECA overproducer strains. We found: a) a normal induction of RECA following UV irradiation of E. coli bacteria overproducing SSB, b) a normal level of SSB in wild type and ssb-1 and ssb-113 mutants either in the absence or in the presence of S.O.S. inducing agents. We confirmed a severe impairment in the induction of RECA in these two mutants after nalidixic acid treatment. Our results suggest that the concentrations of RECA and SSB protein in E. coli are regulated by independent biochemical pathways.

The content of RECA protein, one of the SOS genes product, was determined in a bacterial extract by a two site-radioimmunometric assay. The variation of the RECA concentration after induction by physical or chemical treatments was used as a probe to analyze the SOS response. Relationships between either the number or the nature of DNA lesions and the level of the relative amplification of RECA have been established. The modulation of the recA gene expression is discussed.

A two-site immunoradiometric assay of Escherichia coli recA protein is described ; its sensitivity allows the detection of 0.1 ng of recA protein ; it yields a linear response for amounts of recA protein in the 0.1-7 ng range. It can be directly applied to extracts of Escherichia coli obtained by sonication. Salmonella typhimurium extracts contain some cross-reacting material which share common antigenic determinants with Escherichia coli recA protein but differ from it.

RecA protein was purified from S. typhimurium and its concentration was measured in crude extracts by an immunoradiometric assay. The dose-response relations and the kinetics of recA protein induction following treatment of the cells with ultra-violet light, nalidixic acid, mitomycin C, and cisplatin were studied in E. coli and S. typhimurium. The recA protein amplification was complete in a few hours and was stable for at least 3 hours. Dose-response curves showed a linear region for low doses of all the inducer agents tested. This direct relation between the recA protein level and the amount of inducer agent allows the quantification of the recA protein inducing potency of chemicals. The recA protein amplification was very sensitive to low doses of inducer agents: an UV dose of 0.25 J/M2, 500 ng of NAL or 50 ng of MMC induced a two-fold increase in cellular recA protein content. In addition, the measurement of RecA protein induction did not require the survival of the cells. These observations led us to suggest a new biochemical assay for detecting DNA damaging substances by the direct measurement of the recA protein level following treatment of the cells.