Albany 2001

category image Biomolecular
SUNY at Albany
June 19-23, 2001

Crystal structure of the clamp loader small subunit from Pyrococcus furiosus

In the eukaryotic DNA replication, replication factor C (RFC) is required as the clamp loader at the replication fork upon loading the PCNA, the processivity factor of DNA polymerases, onto nascent DNA strands. RFC catalyzes this loading reaction in an ATP dependent manner probably by opening the PCNA ring. Eukaryotic RFC comprises one large (95-130 kDa) and four distinct small (35-40 kDa) subunits, and all five subunits share sequence similarity referred to as RFC boxes. The archaeal replication systems are considered to be similar to the eukaryotic ones. The RFC from Pyrococcus furiosus is thought to be a complex of RFCS (small, 37 kDa) and RFCL (large, 55 kDa) in a ratio 4 : 1 or 2. Recent biochemical analyses of the clamp loader showed that its functions are similar to those of eukaryotic complexes (1). To elucidate the molecular mechanisms of the PCNA loading reaction at atomic level, we have determined the crystal structure of the RFCS at 2.8 angstrom resolution. The RFCS assembles in a hexamer with a pseudo dimer-of-trimer symmetry, which is most likely to be a dislocated form of a 6-fold ring observed in EM analysis (2). The crescent shape formed by three domains of the RFCS subunit overall resembles that of the delta-prime subunit of E. coli polymerase III holo-enzyme (3). The N-terminal two domains form a nucleotide binding site and share structural similarity with members of the AAA+ superfamily. Together with results from mutational analyses, the consideration of the subunit organization suggests that the obtained crystal structure could include a part of the active holo-complex composed of the RFCS and the RFCL.

References and Footnotes
  1. Cann, I.K.O., Ishino, S., Yuasa, M., Daiyasu, H., Toh, H., and Ishino, Y. (2001) Submitted.
  2. Mayanagi, K., Miyata, T., Oyama, T., Ishino, Y., and Morikawa, K. (2001) Submitted.
  3. Guenther, B., Onrust, R., Sail, A., O'Donnell, M., and Kuriyan, J. (1997). Cell 91, 335-345.

Takuji Oyama(1), Yoshizumi Ishino(2), Isaac K. O. Cann(2) and Kosuke Morikawa(1)

Department of Structural Biology(1), Department of Molecular Biology(2), Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita-City, Osaka 565-0874, Japan.
E-mail: chicago@beri.co.jp, Phone: +(81) 6 6872 8201, FAX: +(81) 6 6872 8219