Purification and properties of an accessory protein for DNA polymerase alpha/primase.
A protein that stimulates DNA polymerase alpha/primase many-fold on unprimed poly(dT) was purified to homogeneity from extracts of cultured mouse cells. The protein contains polypeptides of approximately 132 and 44 kDa, and the total molecular mass of 150 kDa calculated from Stokes radius (54 A) and sedimentation coefficient (6.7 S) indicates that it contains one each of the two subunits. The purified "alpha accessory factor" (AAF) also stimulates DNA polymerase alpha/primase in the self-primed reaction with unprimed single-stranded DNA. In addition to these effects on the coordinate activities of DNA polymerase alpha and DNA primase, stimulatory effects were also demonstrated separately on both the polymerase and primase activities of the enzyme complex. However, there was no stimulation with DNase-treated ("activated") DNA under normal conditions for assay of DNA polymerase alpha. The stimulatory activity of mouse AAF is highly specific for DNA polymerase alpha/primase; no effect was observed with mouse DNA polymerases beta, gamma, or delta, nor with retroviral, bacteriophage, or bacterial DNA polymerases. Mouse AAF stimulated human DNA polymerase alpha/primase with several different templates, similar to results with the mouse enzyme. However, it had very little effect on the DNA polymerase/primase from either Drosophila embryo or from yeast.
Goulian
M
M
Department of Medicine, University of California, San Diego, La Jolla 92093-0613.
Heard
C J
CJ
Grimm
S L
SL
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D019915:DNA Primase; D017886:Durapatite; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D006882:Hydroxyapatites; D007700:Kinetics; D007939:Leukemia L1210 / Q000201:enzymology; D051379:Mice; D011071:Poly T / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D013698:Templates, Genetic
D006882:Hydroxyapatites; D011506:Proteins; D011071:Poly T; D017886:Durapatite; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases