Human-Saccharomyces cerevisiae proliferating cell nuclear antigen hybrids: oligomeric structure and functional characterization using in vitro DNA replication
| Autor: | A, Ola, S, Waga, V, Ellison, B, Stillman, M, McGurk, I M, Leigh, N H, Waseem, A, Waseem |
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| Rok vydání: | 2000 |
| Předmět: |
Protein Folding
DNA Complementary Saccharomyces cerevisiae Proteins Recombinant Fusion Proteins Immunoblotting Saccharomyces cerevisiae Thymus Gland Models Biological Minor Histocompatibility Antigens Proliferating Cell Nuclear Antigen Animals Humans Replication Protein C DNA Polymerase III Adenosine Triphosphatases Homeodomain Proteins Binding Sites Dose-Response Relationship Drug DNA Protein Structure Tertiary DNA-Binding Proteins Repressor Proteins Cross-Linking Reagents Proto-Oncogene Proteins c-bcl-2 Mutation Chromatography Gel Mutagenesis Site-Directed Cattle Electrophoresis Polyacrylamide Gel Dimerization Plasmids |
| Zdroj: | The Journal of biological chemistry. 276(13) |
| ISSN: | 0021-9258 |
| Popis: | The proliferating cell nuclear antigen (PCNA) is a highly conserved protein required for the assembly of the DNA polymerase delta (pol delta) holoenzyme. Because PCNAs from Saccharomyces cerevisiae and human do not complement each other using in vitro or in vivo assays, hybrids of the two proteins would help identify region(s) involved in the assembly of the pol delta holoenzyme. Two mutants of human PCNA, HU1 (D21E) and HU3 (D120E), and six hybrids of human and S. cerevisiae PCNA, HC1, HC5, CH2, CH3, CH4, and CH5, were prepared by swapping corresponding regions between the two proteins. In solution, all PCNA assembled into trimers, albeit to different extents. These PCNA variants were tested for stimulation of pol delta and in vitro replication of M13 and SV40 DNA as well as to stimulate the ATPase activity of replication factor C (RF-C). Our data suggest that in addition to the interdomain connecting loop and C terminus, an additional site in the N terminus is required for pol delta interaction. PCNA mutants and hybrids that stimulated pol delta and RF-C were deficient in M13 and SV40 DNA replication assays, indicating that PCNA-induced pol delta stimulation and RF-C-mediated loading are not sufficient for coordinated DNA synthesis at a replication fork. |
| Databáze: | OpenAIRE |
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