Purification and characterization of a yeast DNA polymerase alpha complex with associated primase, 5'-->3' exonuclease, and DNA-dependent ATPase activities.
We have purified a multimeric form of yeast DNA polymerase alpha with DNA polymerase, primase, 5'-->3' exonuclease, and single-stranded (ss) DNA-dependent ATPase activities to near-homogeneity. The molecular mass of complex was 650 kDa with subunits ranging in sizes from 30 to 180 kDa. The alpha-subunit of the complex could be detected by DNA polymerase alpha antibody. No cross-reactivity of polypeptides within the complex was observed with antibodies directed against polymerase delta or epsilon. The multimeric polymerase alpha could be selectively inhibited by p-n-butylphenyl-dGTP (I50 of approximately 0.2 microM), p-n-butylanilino-dATP (I50 of 1.3 microM), and aphidicolin (I50 of 2.5 micrograms/mL). The complex synthesized RNA primers on various ssDNA templates and rapidly elongated these primers into nascent DNA fragments in the presence of required deoxynucleotides. It has a strong 5'-->3' exonuclease activity. In addition, the complex hydrolyzed both ATP and dATP in a ssDNA-dependent manner. Thus, the multiprotein complex of DNA polymerase alpha had multiple activities (primase, polymerase, and ATPase) which could act concertedly to synthesize primers and elongate the primers to nascent DNA fragments in the lagging strand of the fork.
Biswas
E E
EE
Department of Pediatrics, University of Maryland School of Medicine, Baltimore 21201.
Chen
P H
PH
Gray
W
W
Li
Y H
YH
Ray
S
S
Biswas
S B
SB
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000096:biosynthesis; D004257:DNA Polymerase II / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D019915:DNA Primase; D004277:DNA, Single-Stranded / Q000378:metabolism / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D046912:Multiprotein Complexes; D009841:Oligonucleotides / Q000096:biosynthesis; D012313:RNA / Q000096:biosynthesis; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D004277:DNA, Single-Stranded; D046911:Macromolecular Substances; D046912:Multiprotein Complexes; D009841:Oligonucleotides; D012313:RNA; D000255:Adenosine Triphosphate; D004247:DNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D000251:Adenosine Triphosphatases