Mutational analysis of Pyrococcus furiosus replication factor C based on the three-dimensional structure

Sonoko Ishino, Takuji Oyama, Mihoko Yuasa, Kosuke Morikawa, Yoshizumi Ishino

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

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-Å resolution . Using the information from the determined tertiary structure, we prepared several mutations in RFCS and biochemically characterized them. Truncation of the C-terminal α-helix (α16) 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.

Original languageEnglish
Pages (from-to)169-175
Number of pages7
JournalExtremophiles
Volume7
Issue number3
DOIs
Publication statusPublished - Jun 1 2003

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Medicine

Fingerprint Dive into the research topics of 'Mutational analysis of Pyrococcus furiosus replication factor C based on the three-dimensional structure'. Together they form a unique fingerprint.

Cite this