A 21S enzyme complex from HeLa cells that functions in simian virus 40 DNA replication in vitro.
A sedimentable complex of enzymes for DNA synthesis was partially purified from the combined low-salt nuclear extract-postmicrosomal supernatant solution of HeLa cell homogenates by poly(ethylene glycol) precipitation in the presence of 2 M KCl, discontinuous gradient centrifugation, Q-Sepharose chromatography, and velocity gradient centrifugation. In addition to the previously described 640-kDa multiprotein DNA polymerase alpha-primase complex [Vishwanatha et al. (1986) J. Biol. Chem. 261, 6619-6628], the enzyme complex also has associated topoisomerase I, DNA-dependent ATPase, RNase H, DNA ligase, a simian virus 40 origin recognition, dA/dT sequence binding protein [Malkas & Baril (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 70-74], and proliferating cell nuclear antigen. Essentially all of the T antigen dependent simian virus 40 in vitro replication activity in the combined nuclear extract-postmicrosomal supernatant solution resides with the sedimentable complex of enzymes for DNA synthesis. Sedimentation analysis on a 10-35% glycerol gradient in the presence of 0.5 M KCl indicates that the enzyme complex is 21S. The associated enzymes for DNA synthesis and in vitro simian virus 40 replication activity cofractionate throughout the purification of the 21S complex. The DNA polymerase and in vitro simian virus 40 replication activities are both inhibited by monoclonal antibody (SJK 132-20) to human DNA polymerase alpha and by 5-10 microM butylphenyl-dGTP, indicating that the association of DNA polymerase alpha with the 21S enzyme complex is essential for the initiation of SV40 DNA replication in vitro.
Malkas
L H
LH
Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Hickey
R J
RJ
Li
C
C
Pedersen
N
N
Baril
E F
EF
D000251:Adenosine Triphosphatases / Q000302:isolation & purification; D001483:Base Sequence; D002847:Chromatography, Agarose; D004265:DNA Helicases; D011088:DNA Ligases / Q000302:isolation & purification; D004257:DNA Polymerase II / Q000302:isolation & purification; D019915:DNA Primase; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000302:isolation & purification; D004722:Endoribonucleases / Q000302:isolation & purification; D006367:HeLa Cells / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000502:physiology*; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification; D016914:Ribonuclease H; D013539:Simian virus 40 / Q000235:genetics / Q000502:physiology*; D014779:Virus Replication
D009097:Multienzyme Complexes; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II; D004722:Endoribonucleases; D016914:Ribonuclease H; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D004264:DNA Topoisomerases, Type I; D011088:DNA Ligases