Stimulation and inhibition of deoxyribonucleic acid nucleotidyltransferase by oligodeoxyribonucleotides

Publisher Website
Google Scholar
Abstract
Soluble extracts prepared directly from osmotically disrupted suspensions of Landschutz ascites-tumour cells contained less deoxyribonuclease activity than extracts prepared in the presence of potassium chloride-phosphate buffer. Increased deoxyribonuclease activities were accompanied by increased deoxyribonucleic acid nucleotidyltransferase activities. Prolonged extraction procedures yielded preparations containing a factor that diminished these increased activities of deoxyribonuclease and nucleotidyltransferase. Acid fractionation removed much of the deoxyribonuclease activity from the soluble extracts and purified the preparation sixfold with respect to the nucleotidyltransferase. The lag phase observed under standard conditions of assay of the transferase in soluble extracts and in the partially purified fraction was eliminated by addition of certain 5[image]-phosphorylterminal oligonucleotides from a pancreatic-deoxyribonuclease acid; also, the extent of synthesis of polydeoxyribonucleotide was increased, and, assuming that the product was derived from oligonucleotides and deoxyribonucleoside triphosphates, the synthesis amounted to 74% of the deoxyribonucleic acid primer. The nucleotidyltransferase was inhibited and the lag phase extended by 3[image]-phosphoryl-terminal oligonucleotides from a splenic-deoxyribonuclease digest of deoxyribonucleic acid. The compounds obtained by removal of terminal phos-phoryl groups from the 3[image]- and 5[image]-phosphoryl-terminal oligonucleotides by bacterial alkaline phosphatase abolished the lag phase, and they supported synthesis at a level similar to that observed under standard assay conditions. It was concluded that 5[image]-phosphoryl-terminal oligonucleotides associate by specific hydrogen bonding with single-stranded regions of the deoxyribonucleic acid primer and stimulate synthesis of polydeoxyribonucleotide by providing 3[image]-hydroxyl groups to initiate polymerization along the adjacent single-stranded template. The 5[image]-phosphoryl-terminal group on these oligonucleotides is partly responsible for the stimulation observed. Inhibition by 3[image]-phosphoryl-terminal oligonucleotides is attributable to competition with 3[image]-hydroxyl groups formed on the deoxyribonucleic acid primer by deoxyribonuclease during the nucleotidyltransferase assay, and the lag phase observed under standard conditions is a period of production of stimulatory end groups in the deoxyribonucleic acid primer.