Reconstitution of complete SV40 DNA replication with purified replication factors

Waga, S., Bauer, G., Stillman, B. (April 1994) Reconstitution of complete SV40 DNA replication with purified replication factors. Journal of Biological Chemistry, 269 (14). pp. 10923-34. ISSN 0021-9258



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.

Item Type: Paper
Uncontrolled Keywords: Animals Cattle DNA Ligases metabolism DNA Replication DNA Viral biosynthesis DNA-Binding Proteins metabolism Exonucleases metabolism Homeodomain Proteins Humans Nuclear Proteins metabolism Proto-Oncogene Proteins c-bcl-2 Replication Factor C Repressor Proteins Saccharomyces cerevisiae Proteins Simian virus 40 genetics
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > DNA replication
biotechnology > chromatography > protein purification
CSHL Authors:
Communities: CSHL labs > Stillman lab
Highlight: Stillman, Bruce W.
Depositing User: CSHL Librarian
Date: 8 April 1994
Date Deposited: 17 Feb 2012 20:31
Last Modified: 20 Jun 2017 19:50
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