Characterization of DNA polymerase-alpha/primase complex from developing embryonic chicken brains.
DNA polymerase-alpha and primase activities present in a complex, have been isolated, partially purified, and characterized from embryonic chicken brain. DNA polymerase-alpha activity, characterized by its sensitivity to N-ethyl-maleimide, high sedimentation coefficient (11.3 S), and acidic isoelectric point (5-5.5) was found in all embryonic ages. Primase activity, the enzyme responsible for the initiation of DNA synthesis, co-sedimented with DNA polymerase-alpha activity on a continuous glycerol velocity gradient. A complex containing both DNA polymerase-alpha and primase activities was isolated by DE-23 cellulose column chromatography of cell-free extracts of different embryonic ages of chicken brain. In addition to the primase complexed with DNA polymerase-alpha, a free primase activity was isolated by DE-23 cellulose column chromatography of an ammonium sulfate (0-45%; w/v) precipitated fraction of embryonic chicken brain cell-free extract. DNA polymerase-alpha activity from developing chicken brains in the embryonic stage was purified by immuno-affinity column chromatography. Of all the single-stranded DNA templates tested, primase activity was found to be maximally active with poly dC. Primase activity was not inhibited by a high concentration of alpha-amanitin. The results obtained may provide insight into further understanding of regulation of chromosomal DNA replication in developing tissues.
Ray
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Department of Chemistry and Biochemistry, University of Notre Dame, IN 46556.
Kelley
T J
TJ
Fan
L
L
Basu
S
S
D000818:Animals; D001921:Brain / Q000196:embryology / Q000201:enzymology*; D002642:Chick Embryo; D019915:DNA Primase; D004261:DNA Replication; D012316:RNA Nucleotidyltransferases / Q000032:analysis*
D019915:DNA Primase; D012316:RNA Nucleotidyltransferases