DNA polymerase alpha-DNA primase from human lymphoblasts.
The DNA polymerase alpha-DNA primase complex from the human lymphoblast line HSC93 has been enriched to near homogeneity by using an immunoaffinity purification protocol which was developed earlier for the purification of the calf thymus enzyme (Nasheuer, H.-P. and Grosse, F. (1987) Biochemistry 26, 8458-8466). Immunoaffinity purified polymerase-primase from human cells consisted of four subunits displaying molecular weights of 195,000 and 180,000 for the DNA synthesizing alpha-subunit, of 68,000 for the beta-subunit, and of 55,000 and 48,000 for the primase-carrying gamma- and delta-subunit, respectively. The isoelectric pH values for the individual subunits were estimated from non-equilibrium pH gradients to be between 5.9 and 5.7 for the alpha-subunit, at 5.5 for the beta-subunit, and at 7.5 and 8.0 for the gamma- and delta-subunit, respectively. The purified polymerase-primase converted single-stranded phi X174 DNA into the double-stranded form in a primase-initiated reaction. During this process, 3-10 RNA primers were formed. RNA primers were about 11 nucleotides long. Elongation of existing RNA primers by the human polymerase-primase was semi-processive; following primer binding the DNA polymerase continuously incorporated 20 to 50 nucleotides, then it dissociated from the template DNA.
Bialek
G
G
Department of Chemistry, Max-Planck-Institute for Experimental Medicine, Göttingen, F.R.G.
Nasheuer
H P
HP
Goetz
H
H
Behnke
B
B
Grosse
F
F
D010584:Bacteriophage phi X 174 / Q000235:genetics; D002384:Catalysis; D002460:Cell Line; D002846:Chromatography, Affinity; D019915:DNA Primase; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008214:Lymphocytes / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*
D004279:DNA, Viral; D046911:Macromolecular Substances; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases