Studies on the Synthesis of Deoxyribonucleic Acid: II. Studies with Biologically Active Templates and the Stimulation of Synthesis by Oligonucleotides

Abstract

Abstract When purified T5-induced deoxyribonucleic acid polymerase is incubated with transforming deoxyribonucleic acid from Bacillus subtilis, there is no detectable loss of transforming ability. By using 5-bromodeoxyuridine triphosphate triphosphate as a density marker and 32P-labeled transforming deoxyribonucleic acid as template, it was shown that after two rounds of synthesis, fractions isolated from the regions of a density gradient equivalent to deoxyribonucleic acid fully substituted with bromuracil and containing no detectable 32P had low but significant transforming activity. When deoxyribonuclease digests are added to incubation mixtures containing single stranded deoxyribonucleic acid and purified T5-induced polymerase, there is a marked stimulation of both the rate and yield. Digests prepared from the same type of deoxyribonucleic acid used as template are more effective than digests of heterologous nucleic acid. From analysis of sedimentation patterns in alkaline sucrose, and from experiments involving gel filtration, it has been found that oligonucleotides present in the digest are incorporated into the product in covalent linkage and probably act as initiators for deoxyribonucleic acid synthesis in this system.