Purification of a calf thymus DNA-dependent adenosinetriphosphatase that prefers a primer-template junction effector.
A purification procedure has been developed that resolves four chromatographically distinct DNA-dependent ATPase activities from calf thymus tissue. One of these activities has been purified to a nearly homogeneous protein, as judged by polyacrylamide gel electrophoresis. This protein has a specific activity of 18 mumol of ATP hydrolyzed per minute per milligram of protein and is active only in the presence of a DNA effector. The DNA-dependent ATPase activity is greatest in the presence of DNA containing a 3'-hydroxyl primer-template junction with a segment of adjacent single strand, i.e., a DNA polymerase substrate. Primer-template effectors that have had the 3'-hydroxyl group eliminated by the addition of a dideoxyribonucleotide are less active as cofactors for ATP hydrolysis than effectors which retain the 3'-hydroxyl group. Other DNAs can serve as cofactors, but with a reduced rate of ATP hydrolysis. DNA cofactors which are single stranded are much more effective at promoting ATPase activity than completely double-stranded cofactors, although the effectiveness of single-stranded DNA decreases as the length of the oligonucleotide decreases. An RNA/DNA hybrid does not promote ATPase activity. These data suggest that ATPase A may be involved in the recognition of primer-template junctions and the elongation phase of DNA synthesis.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D004265:DNA Helicases / Q000302:isolation & purification*; D004256:DNA Polymerase I / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D014176:Protein Biosynthesis; D013329:Structure-Activity Relationship; D013698:Templates, Genetic; D013950:Thymus Gland
D004256:DNA Polymerase I; D000251:Adenosine Triphosphatases; D004265:DNA Helicases