title: Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis.
abstract: We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase ɛ (Pol ɛ), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.
authors.affiliation: Department of Molecular and Cellular Biology, Beckman Research Institute at City of Hope, Duarte, California, United States of America.
authors.forename: Caroline M
authors.initials: CM
authors.lastname: Li
authors.affiliation: Department of Molecular Medicine, Beckman Research Institute at City of Hope, Duarte, California, United States of America.
authors.forename: Yunan
authors.initials: Y
authors.lastname: Miao
authors.affiliation: Department of Molecular and Cellular Biology, Beckman Research Institute at City of Hope, Duarte, California, United States of America.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Lingeman
authors.affiliation: Department of Molecular Medicine, Beckman Research Institute at City of Hope, Duarte, California, United States of America.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Hickey
authors.affiliation: Department of Molecular and Cellular Biology, Beckman Research Institute at City of Hope, Duarte, California, United States of America.
authors.forename: Linda H
authors.initials: LH
authors.lastname: Malkas
mesh_terms: D000957:Antigens, Viral, Tumor / Q000235:genetics; D002457:Cell Extracts / Q000737:chemistry; D045744:Cell Line, Tumor; D004256:DNA Polymerase I / Q000302:isolation & purification; D004257:DNA Polymerase II / Q000302:isolation & purification; D004258:DNA Polymerase III / Q000302:isolation & purification; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase / Q000032:analysis* / Q000302:isolation & purification*; D004586:Electrophoresis / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D009097:Multienzyme Complexes / Q000032:analysis* / Q000302:isolation & purification*; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D018741:Replication Origin / Q000235:genetics; D051716:Replication Protein A / Q000302:isolation & purification; D051818:Replication Protein C / Q000302:isolation & purification; D013539:Simian virus 40 / Q000235:genetics; D000099272:Replisomes
chemical_list: D000957:Antigens, Viral, Tumor; D002457:Cell Extracts; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D004264:DNA Topoisomerases, Type I; D004259:DNA-Directed DNA Polymerase; D009097:Multienzyme Complexes; D018809:Proliferating Cell Nuclear Antigen; D051716:Replication Protein A; D051818:Replication Protein C; D000099272:Replisomes