The DNA synthesizing subunit of polymerase-primase from calf thymus.
The DNA synthesizing subunit (alpha-subunit) of DNA polymerase-alpha from calf thymus was separated from the other three subunits by immunoaffinity chromatography. The enzymatic properties of the alpha-subunit were characterized and compared with those of the four-subunit complex. Free alpha-subunit behaved in many respects like the four-subunit polymerase-primase. It was inhibited by aphidicolin and butylanilino-deoxyATP and catalyzed DNA synthesis on both gapped duplex DNA as well as primed single-stranded DNA with a preference of gapped DNA. The alpha-subunit is a quasi-processive enzyme with a processivity for about 9 nucleotides incorporated per single primer binding event. This is 2-fold lower than the processivity of the four-subunit complex. Despite this moderate processivity, free alpha-subunit was able to synthesize long stretches of DNA on singly primed natural psi X174am16 DNA. The accuracy of DNA synthesis of the free alpha-subunit was determined by using the psi X174am16 reversion assay to be 1 error per 50,000 nucleotides incorporated. An in vitro accuracy of 1 error in 54,000 nucleotides incorporated was measured in parallel for the four-subunit complex. Thus, the smaller subunits do not contribute to the overall accuracy of DNA polymerase-alpha. Consistent with this result is the observation that the polymerase to 3'----5'-exonuclease ratio was less than 1 to 2,500,000. Therefore, there is no evidence for the action of a cryptic proofreading activity with the alpha-subunit of DNA polymerase-alpha of mammalian origin.
Bialek
G
G
Max-Planck-Institut für Experimentelle Medizin, Abteilung Chemie, Göttingen, Federal Republic of Germany.
Grosse
F
F
D000818:Animals; D010584:Bacteriophage phi X 174 / Q000235:genetics; D002417:Cattle; D019915:DNA Primase; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics; D007700:Kinetics; D046911:Macromolecular Substances; D011067:Poly dA-dT; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D013698:Templates, Genetic; D013950:Thymus Gland / Q000201:enzymology*
D004279:DNA, Viral; D046911:Macromolecular Substances; D011067:Poly dA-dT; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases