DNA primase-DNA polymerase alpha assembly from mouse FM3A cells. Purification of constituting enzymes, reconstitution, and analysis of RNA priming as coupled to DNA synthesis.
The mouse DNA primase-DNA polymerase alpha complex can be resolved with buffer containing 50% ethylene glycol (Suzuki, M., Enomoto, T., Hanaoka, F., and Yamada, M. (1985) J. Biochem. (Tokyo) 98, 581-584). The dissociated primase and DNA polymerase alpha have been purified sufficiently that there was no cross-contamination with each other. By the use of thus isolated DNA primase and DNA polymerase alpha in addition to DNA primase-DNA polymerase alpha complex, we have studied primer RNA synthesis and DNA elongation separately as well as the coupled reaction of the initiation and elongation of DNA chains. In the absence of deoxyribonucleoside triphosphates, the isolated primase synthesized oligoribonucleotides of an apparent length of 7-11 nucleotides (monomeric oligomer) and multiples of a modal length of 9-10 nucleotides (multimeric oligomer) and fd phage single-stranded circular DNA. Monomeric and dimeric oligomers were synthesized processively, and trimeric and larger oligomers were produced by repeated cycles of processive synthesis. The primase complexed with DNA polymerase alpha mainly synthesized monomeric and a small amount of dimeric oligomers. In the presence of deoxyribonucleoside triphosphates at concentrations above 10 microM, the DNA primase-DNA polymerase alpha complex exclusively synthesized monomeric oligomers only, which were utilized as primers for DNA synthesis. On the other hand, the products synthesized by the isolated primase were qualitatively unchanged as compared with those synthesized in the absence of DNA precursors. When the synthesis of oligomers by the isolated primase was coupled with DNA elongation by the addition of the primase-free DNA polymerase alpha, the synthesis of dimeric oligomers was inhibited as a result of efficient DNA elongation from monomeric oligomers.
Suzuki
M
M
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Enomoto
T
T
Masutani
C
C
Hanaoka
F
F
Yamada
M
M
Ui
M
M
D000818:Animals; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002848:Chromatography, DEAE-Cellulose; D019915:DNA Primase; D004261:DNA Replication; D051379:Mice; D009838:Oligodeoxyribonucleotides / Q000096:biosynthesis; D012316:RNA Nucleotidyltransferases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D013698:Templates, Genetic
D009838:Oligodeoxyribonucleotides; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases