Further purification and characterization of a multienzyme complex for DNA synthesis in human cells.
The 21 S complex of enzymes for DNA synthesis in the combined low salt nuclear extract-post microsomal supernatant from HeLa cells [Malkas et al. (1990) Biochemistry 29:6362-6374] was purified by poly (ethylene glycol) precipitation, Q-Sepharose chromatography, Mono Q Fast Protein Liquid Chromatography (FPLC), and velocity gradient centrifugation. The procedure gives purified enzyme complex at a yield of 45%. The 21 S enzyme complex remains intact and functional in the replication of simian virus 40 DNA throughout the purification. Sedimentation analysis showed that the 21 S enzyme complex exists in the crude HeLa cell extract and that simian virus 40 in vitro DNA replication activity in the cell extract resides exclusively with the 21 S complex. The results of enzyme and immunological analysis indicate that DNA polymerase alpha-primase, a 3',5' exonuclease, DNA ligase I, RNase H, and topoisomerase I are associated with the purified enzyme complex. Denaturing polyacrylamide gel electrophoresis of the purified enzyme complex showed the presence of about 30 polypeptides in the size range of 300 to 15 kDa. Immunofluorescent imaging analysis, with antibodies to DNA polymerase alpha,beta and DNA ligase I, showed that polymerase alpha and DNA ligase I are localized to granular-like foci within the nucleus during S-phase. In contrast, DNA polymerase beta, which is not associated with the 21 S complex, is diffusely distributed throughout the nucleoplasm.
Li
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Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Cao
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Wang
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Baril
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EF
D001483:Base Sequence; D002467:Cell Nucleus / Q000201:enzymology; D011232:Chemical Precipitation; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000302:isolation & purification / Q000378:metabolism; D004256:DNA Polymerase I / Q000302:isolation & purification / Q000378:metabolism; D004257:DNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000302:isolation & purification / Q000378:metabolism; D004279:DNA, Viral / Q000096:biosynthesis*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism*; D011092:Polyethylene Glycols; D016914:Ribonuclease H / Q000302:isolation & purification / Q000378:metabolism; D016196:S Phase; D013539:Simian virus 40 / Q000235:genetics; D014461:Ultracentrifugation; D014779:Virus Replication
D004279:DNA, Viral; C000607892:LIG1 protein, human; D009097:Multienzyme Complexes; D011092:Polyethylene Glycols; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D016914:Ribonuclease H; D004264:DNA Topoisomerases, Type I; D011088:DNA Ligases; D000072481:DNA Ligase ATP