PROCESSIVITY OF ESCHERICHIA-COLI AND RAT-LIVER MITOCHONDRIAL URACIL-DNA GLYCOSYLASE IS AFFECTED BY NACL CONCENTRATION

Escherichia coli uracil-DNA glycosylase was shown to catalyze the hydrolysis of a site-specific uracil residue from a defined single-stranded oligonucleotide (25-mer). With duplex 25-mer, the rate of uracil removal from double-stranded DNA containing a U . G mispair was similar to-2-fold greater than a U . A base pair. The mechanism by which E. coli and rat liver mitochondrial uracil-DNA glycosylase located sequential uracil residues within double-stranded DNA was investigated. Two concatemeric polynucleotide substrates were constructed by ligation of homologous 5'-end P-32-labeled 25-mer double-stranded oligonucleotides that contained either a site-specific U . G or U . A target site at intervals of 25 nucleotides along one strand of the DNA. Reaction of uracil-DNA glycosylase with these concatemeric DNAs, followed by alkaline hydrolysis of the resultant AP-sites, would produce predominantly [P-32]25-mer products, if a processive mechanism was used to locate successive uracil residues, or oligomeric multiples of [P-32]25-mer, if a distributive mode was exhibited. Both the bacterial and the mitochondrial enzymes were found to act processively on U . A- and U . G-containing DNA in the absence of NaCl, based on the initial rate of 25-mer produced relative to the total amount of uracil excised. Approximately 50% of the total uracil excised resulted in the release of 25-mer product. The addition of NaCl (greater than or equal to 50 mM) caused reduced processivity on both U . A- and U . G-containing DNA substrates. The mode of action of uracil-DNA glycosylase was Very similar to that observed for the EcoRI endonuclease cleavage of restriction sites contained in the same DNA substrate which was used as a positive control.