Analysis of the mechanism of ATP stimulation of calf thymus DNA alpha-polymerase.
Biochemical kinetic analyses of the ATP stimulation of the A2 form of calf DNA alpha-polymerase show that when DNA or primer termini are the variable substrates, maximum reaction velocity is independent of ATP concentration. When dNTP concentration is the variable substrate, the apparent Km is invariant with ATP. Such results indicate that the increase in the synthetic rate caused by ATP results from an improvement in synthesis initiation at primer termini. The effect of ATP on the DNA binding affinity of alpha-A2-polymerase was examined by using column chromatography. Passage of the polymerase through native DNA-cellulose at 70 mM ionic strength resulted in 40% binding of the enzyme. In the presence of 4 mM ATP, binding increased to 80%. In both cases, the bound polymerase could be eluted by a 370 mM ionic strength wash. An elution profile similar to that observed in the absence of ATP was obtained with 0.1 mM ATP, 4 mM GTP, or 4 mM each of the nonhydrolyzable ATP analogues adenyl-5'-yl imidodiphosphate or adenosine 5'-O-(3-thiotriphosphate). These results suggest that hydrolysis of the gamma-phosphate occurs at millimolar levels of ATP and leads to a higher affinity of polymerase for DNA. To distinguish the effects of ATP on RNA priming from those on DNA synthesis, products synthesized processively by alpha-A2-polymerase were sized by gel filtration. Results indicate that essentially all products made on a gapped fd replicative form template in the presence of four dNTPs and 4 mM ATP result from the extension of preexisting DNA primers.(ABSTRACT TRUNCATED AT 250 WORDS)
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D004247:DNA / Q000031:analogs & derivatives / Q000378:metabolism; D004257:DNA Polymerase II / Q000378:metabolism*; D019915:DNA Primase; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D013698:Templates, Genetic; D013950:Thymus Gland / Q000201:enzymology*
C033190:DNA-cellulose; D000266:Adenylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D002482:Cellulose; D004247:DNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II