Purification and characterization of mouse DNA polymerase alpha devoid of primase activity.
A simple method was developed for the isolation of primase-free DNA polymerase-alpha from the DNA polymerase-alpha-primase complex of mouse FM3A cells. The polymerase was separated from primase subunits by chromatography on a single-stranded DNA-cellulose column in the presence of 50% etylene glycol. The primase-free DNA polymerase-alpha contained two polypeptides with molecular masses of 180,000 and 68,000. Analysis of the DNA products with poly(dA)-oligo(dT)10 as template-primer revealed that both primase-free DNA polymerase-alpha and the DNA polymerase-alpha-primase complex predominantly synthesized short DNA with less than 30 nucleotides, but that the DNA polymerase-alpha-primase complex also synthesized some longer DNA with more than 300-400 nucleotides.
Takada-Takayama
R
R
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Suzuki
M
M
Enomoto
T
T
Hanaoka
F
F
Ui
M
M
D000818:Animals; D002460:Cell Line; D002482:Cellulose / Q000031:analogs & derivatives; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004247:DNA; D004257:DNA Polymerase II / Q000302:isolation & purification* / Q000378:metabolism; D019915:DNA Primase; D004261:DNA Replication; D007700:Kinetics; D046911:Macromolecular Substances; D008325:Mammary Neoplasms, Experimental / Q000201:enzymology; D051379:Mice; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification* / Q000378:metabolism
C033190:DNA-cellulose; D046911:Macromolecular Substances; D002482:Cellulose; D004247:DNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II