Mutational analysis of Pyrococcus furiosus replication factor C based on the three-dimensional structure.
In eukaryotic DNA replication, replication factor C (RFC) acts as a "clamp loader" that loads PCNA onto a primed DNA template in an ATP-dependent manner. Proteins with functions essentially identical to that of RFC exist in Archaea. We have determined the crystal structure of the small subunit (RFCS) of Pyrococcus furiosus RFC at 2.8-A resolution. Using the information from the determined tertiary structure, we prepared several mutations in RFCS and biochemically characterized them. Truncation of the C-terminal alpha-helix (alpha16) causes a failure in RFCS oligomerization and a loss of the stimulating activity for the PCNA-dependent DNA synthesis by DNA polymerases. The site-directed reduction of the negative charges at the center part of the RFCS complex affected the stability of the RFC-PCNA interaction and reduced the clamp-loading activity. These results contribute to our general understanding of the structure-function relationship of the RFC molecule for the clamp-loading event.
Ishino
Sonoko
S
Department of Molecular Biology, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, 565-0874 Suita, Osaka, Japan.
Oyama
Takuji
T
Yuasa
Mihoko
M
Morikawa
Kosuke
K
Ishino
Yoshizumi
Y
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D015153:Blotting, Western; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D008957:Models, Genetic; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011233:Precipitin Tests; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D020027:Pyrococcus furiosus / Q000235:genetics*; D051818:Replication Protein C; D013329:Structure-Activity Relationship
D004268:DNA-Binding Proteins; D000255:Adenosine Triphosphate; D004259:DNA-Directed DNA Polymerase; D000251:Adenosine Triphosphatases; D051818:Replication Protein C