NUCLEIC ACID MODIFICATION WITH SPECIFIC ENDONUCLEASES

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The long-term objectives are to characterize useful nucleases and other extracellular enzymes from the marine bacterial genus Alteromonas. The health relatedness of the project lies in the use of these enzymes in genetic engineering-related manipulations of nucleic acids, for which the nucleases have already proven useful in connection with the cloning and expression in E. coli of such medically significant proteins as insulins and interferons. The nucleases, which exist in at least two molecularly and kinetically distinct forms and possess activities against single-stranded DNA, covalent lesions and other distortions in duplex DNA and the termini of linear duplex DNA, are to be further characterized with respect to the dependence of catalytic behavior on a number of environmental variables. Kinetic and reaction mechanisms studies to be done include characterization of the intermediate and final products of digestion of DNA, the elucidation of the directionality and processivity of the activities on linear duplex DNA and single stranded DNAs, the effects of 2',3'-dideoxy terminal nucleotides and of nucleotide analogs bearing a sulfur atom on the Alpha-phosphate on the exonuclease activity when such groups are present at the termini, measurement of the kinetics of conversion of nicked circular to linear duplex DNA and the determination of the location of the site of cleavage in DNA containing a pre-existing strand break to the site of the initial break. A model for the known generation of ligatable fragments bearing single-stranded termini after partial degradation of linear duplex DNAs will be tested, which is expected to lead to a general method for the detection of palindromic sequences in duplex DNAs. The kinetics of cleavage in nonsupercoiled closed circular DNA reacted with a metabolite of the carcinogen benzo[a]pyrene will be determined. One species of the nuclease can apparently be derived from the other by proteolysis and this is to be confirmed and observed intermediates examined. Determination of whether the nuclease cleaves at potential cruciform structures in supercoiled DNA for which this has been shown to be the case for other single-strand-specific nucleases will be done. Attempts to clone the gene(s) for the nuclease will be made, using as first approaches simple methods developed in this laboratory to assay for periplasmic or extracellular nucleases. Preliminary characterization of a phosphatase in culture supernatants that has 5'-nucleotidase activity will be undertaken.