Reconstitution of complete SV40 DNA replication with purified replication factors.
The identification and purification of human cell proteins required for the production of form I DNA following DNA replication from the simian virus 40 (SV40) origin is described. Using these proteins, complete SV40 DNA replication was reconstituted with only purified DNA replication factors: SV40 large tumor antigen (TAg), replication protein A (RPA), DNA topoisomerases I and II, DNA polymerase alpha-primase, replication factor C (RFC), the proliferating cell nuclear antigen (PCNA), DNA polymerase delta, maturation factor 1 (MF1), and DNA ligase I. MF1, a 5' to 3' exonuclease and DNA ligase I were both identified as essential components for production of covalently closed circular relaxed (form I) DNA. MF1 is probably the same exonuclease previously shown by others to function during DNA synthesis on artificial DNA templates or in conjunction with DNA polymerase alpha from the SV40 origin. Combined with these previous studies, our results suggest that MF1 functions to remove an RNA primer attached to every Okazaki fragment during lagging strand DNA synthesis. Interestingly, whereas mammalian DNA ligase I functioned in the reconstituted replication system, mammalian DNA ligase III did not substitute and the phage T4 DNA ligase functioned inefficiently, suggesting that DNA ligase I has a specific role as a replicative DNA ligase in eukaryotic cells.
Waga
S
S
Cold Spring Harbor Laboratory, New York 11724.
Bauer
G
G
Stillman
B
B
D000818:Animals; D002417:Cattle; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism; D018398:Homeodomain Proteins; D006801:Humans; D015778:Minor Histocompatibility Antigens; D009687:Nuclear Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D013539:Simian virus 40 / Q000235:genetics*
C096816:BCL2-related protein A1; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C000607892:LIG1 protein, human; C096063:MATA1 protein, S cerevisiae; D015778:Minor Histocompatibility Antigens; D009687:Nuclear Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D005092:Exonucleases; D051818:Replication Protein C; D011088:DNA Ligases; D000072481:DNA Ligase ATP