The subunits of activator 1 (replication factor C) carry out multiple functions essential for proliferating-cell nuclear antigen-dependent DNA synthesis Journal Article
| Authors: | Pan, Z. Q.; Chen, M.; Hurwitz, J. |
| Article Title: | The subunits of activator 1 (replication factor C) carry out multiple functions essential for proliferating-cell nuclear antigen-dependent DNA synthesis |
| Abstract: | p37 and p40 are two cloned gene products of the five-subunit human cellular DNA replication factor activator 1 (A1) protein complex (also called replication factor C). Here, we describe the solubilization, purification, and characterization of these two proteins that were overproduced in Escherichia coli. Using a nitrocellulose filter binding assay, we demonstrated that the purified A1 p37 protein associated with DNA preferentially at the primer terminus, a property resembling that of the A1 complex. We also show that in the presence of relatively high levels of salt, the recombinant p37 protein alone activated DNA polymerase ε but not polymerase δ in catalyzing the elongation of DNA chains. The p40 protein specifically associated with cellular p37 and proliferating-cell nuclear antigen (PCNA) present in HeLa cell cytosolic extract. The addition of purified p40 protein abolished the in vitro polymerase δ-catalyzed DNA elongation reaction dependent on both PCNA and Al. However, this inhibition was reversed by excess polymerase δ, suggesting a specific interaction between the polymerase and the p40 protein. Thus, while p37 binds DNA at the primer end and has a specific affinity for pol ε, p40, which binds ATP, interacts with PCNA and pol δ. These activities are essential for the DNA elongation reactions that lead to the synthesis of leading-strand DNA and the maturation of Okazaki fragments. |
| Keywords: | dna-binding proteins; dna polymerase; dna replication; dna synthesis; protein analysis; gene product; enzyme activation; hela cells; nuclear proteins; molecular cloning; cloning, molecular; dna; kinetics; protein purification; escherichia coli; recombinant proteins; autoantigens; dna structure; adenosine triphosphate; cycline; protein subunit; enzyme mechanism; electrophoresis, polyacrylamide gel; protein dna interaction; proliferating cell nuclear antigen; dna-directed dna polymerase; chromatography, affinity; protein quaternary structure; dna polymerase iii; human; priority journal; article; support, non-u.s. gov't; support, u.s. gov't, p.h.s.; macromolecular systems; dna polymerases δ and ε |
| Journal Title: | Proceedings of the National Academy of Sciences of the United States of America |
| Volume: | 90 |
| Issue: | 1 |
| ISSN: | 0027-8424 |
| Publisher: | National Academy of Sciences |
| Date Published: | 1993-01-01 |
| Start Page: | 6 |
| End Page: | 10 |
| Language: | English |
| DOI: | 10.1073/pnas.90.1.6 |
| PUBMED: | 8093561 |
| PROVIDER: | scopus |
| PMCID: | PMC45588 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 1 March 2019 -- Source: Scopus |
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