Single-stranded-DNA-dependent ATPase from HeLa cells that stimulates DNA polymerase alpha-primase activity: purification and characterization of the ATPase.
A single-stranded DNA-dependent ATPase that cofractionates during the early stages of purification of a multiprotein DNA polymerase alpha complex from HeLa cells has been purified to homogeneity. The ATPase is part of a 16S multienzyme DNA polymerase alpha complex that is fully active in SV40 DNA replication in vitro. The ATPase hydrolyzes ATP to ADP in a reaction that is completely dependent on the presence of DNA. DNA in single-stranded form is strongly preferred as a cofactor, and polydeoxynucleotides with adenine or thymidine residues are highly effective. Glycerol gradient sedimentation showed that the purified ATPase sedimented at an s20,w of 7 S, and polyacrylamide gel electrophoresis under denaturing conditions reveals two polypeptides with relative molecular weights of 83,000 and 68,000. Both of these polypeptides have purine nucleotide binding sites as revealed by photoaffinity cross-linking experiments. ATP binds to the two subunits more efficiently than GTP, and CTP or UTP does not cross-link with the two polypeptides. DNA synthesis catalyzed by purified HeLa cell DNA polymerase alpha-primase is stimulated in the presence of ATPase and ATP at an optimum concentration of 2 mM. Analysis of the DNA product by gel electrophoresis indicates that with poly(dT) but not phage M13 DNA as template the ATPase overcomes a lag and decreases the length of nascent DNA chains synthesized by the DNA polymerase alpha-primase complex.
Vishwanatha
J K
JK
Department of Biochemistry, University of Nebraska Medical Center, Omaha 68198.
Baril
E F
EF
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D000345:Affinity Labels; D003432:Cross-Linking Reagents; D004265:DNA Helicases; D004257:DNA Polymerase II / Q000378:metabolism*; D019915:DNA Primase; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004789:Enzyme Activation; D006367:HeLa Cells / Q000737:chemistry; D006801:Humans; D009097:Multienzyme Complexes / Q000032:analysis; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism; D011089:Polydeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D009097:Multienzyme Complexes; D009363:Neoplasm Proteins; D011089:Polydeoxyribonucleotides; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II; D000251:Adenosine Triphosphatases; D004265:DNA Helicases