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Biomedical subjects

Sonoko Ishino

Publications and source records attributed to Sonoko Ishino.

7 recordsLinked to original sources
PubMed2006

Comprehensive search for DNA polymerase in the hyperthermophilic archaeon, Pyrococcus furiosus.

DNA polymerase activities were scanned in a Pyrococcus furiosus cell extract to identify all of the DNA polymerases in this organism. Three main fractions containingDNA polymerizing activity were subjected to Western blot analyses, which revealed that the main activities in each fraction were derived from three previously identified DNA polymerases. PCNA (proliferating cell nuclear antigen), the sliding clamp of DNA polymerases, did not bind tightly to any of the three DNA polymerases. A primer usage preference was also shown for each purified DNA polymerase. Considering their biochemical properties, the roles of the three DNA polymerases during DNA replication in the cells are discussed.

Archaeal Proteins↗
PubMed2005

Identification of the critical region in replication factor C from Pyrococcus furiosus for the stable complex formation with proliferating cell nuclear antigen and DNA.

Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are accessory proteins essential for processive DNA synthesis. The function of RFC is to load PCNA, a processivity factor of replicative DNA polymerases, onto primed DNA templates. The central hole of the PCNA homo-trimeric ring encircles doublestranded DNA, so that DNA polymerases can operate for DNA synthesis with PCNA along a DNA template. The Pyrococcus furiosus RFC (PfuRFC) consists of a small subunit (RFCS, 37kDa) and a large subunit (RFCL, 55kDa), which show significant sequence identity to the eukaryotic homologs. The C-terminal region of RFCL has an acidic cluster of about 30 amino acids, which consists mainly of glutamic acid residues, and a following basic cluster of 10 amino acids, which consists mainly of lysine residues. These clusters of charged amino acids, which precede the C-terminal consensus sequence, PIP (PCNA interacting protein)-box, are conserved in several archaeal RFCLs. The series of mutant PfuRFC containing the C-terminal deletions in RFCL were constructed. The mutational analyses showed that the charged cluster is not essential for loading of PCNA onto DNA. However, the region containing the basic cluster is important for the stable ternary (RFC-PCNA-DNA) complex formation.

Amino Acid Sequence↗
PubMed2004

The clamp-loading complex for processive DNA replication.

DNA polymerase requires two processing factors, sliding clamps and clamp loaders, to direct rapid and accurate duplication of genomic DNA. In eukaryotes, proliferating cell nuclear antigen (PCNA), the ring-shaped sliding clamp, encircles double-stranded DNA within its central hole and tethers the DNA polymerases onto DNA. Replication factor C (RFC) acts as the clamp loader, which correctly installs the sliding clamp onto DNA strands in an ATP-dependent manner. Here we report the three-dimensional structure of an archaeal clamp-loading complex (RFC-PCNA-DNA) determined by single-particle EM. The three-dimensional structure of the complex, reconstituted in vitro using a nonhydrolyzable ATP analog, reveals two components, a closed ring and a horseshoe-shaped element, which correspond to PCNA and RFC, respectively. The atomic structure of PCNA fits well into the closed ring, suggesting that this ternary complex represents a state just after the PCNA ring has closed to encircle the DNA duplex.

Adenosine Triphosphate↗
PubMed2003

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.

Adenosine Triphosphatases↗
PubMed2003

Intermolecular ion pairs maintain the toroidal structure of Pyrococcus furiosus PCNA.

Two mutant proliferating cell nuclear antigens from the hyperthermophilic archaeon Pyrococcus furiosus, PfuPCNA(D143A) and PfuPCNA(D143A/D147A), were prepared by site-specific mutagenesis. The results from gel filtration showed that mutations at D143 and D147 drastically affect the stability of the trimeric structure of PfuPCNA. The PfuPCNA(D143A) still retained the activity to stimulate the DNA polymerase reaction, but PfuPCNA(D143A/D147A) lost the activity. Crystal structures of the mutant PfuPCNAs were determined. Although the wild-type PCNA forms a toroidal trimer with intermolecular hydrogen bonds between the N- and C-terminal domains, the mutant PfuPCNAs exist as V-shaped dimers through intermolecular hydrogen bonds between the two C-terminal domains in the crystal. Because the mutated residues are involved in the intermolecular ion pairs through their side chains in the wild-type PfuPCNA, these ion pairs seem to play a key role in maintaining the toroidal structure of the PfuPCNA trimer. The comparison of the crystal structures revealed intriguing conformational flexibility of each domain in the PfuPCNA subunit. This structural versatility of PCNA may be involved in the mechanisms for ring opening and closing.

Amino Acid Substitution↗
PubMed2002

Physical interaction between proliferating cell nuclear antigen and replication factor C from Pyrococcus furiosus.

BACKGROUND: Proliferating cell nuclear antigen (PCNA), which is recognized as a DNA polymerase processivity factor, has direct interactions with various proteins involved in the important genetic information processes in Eukarya. We determined the crystal structure of PCNA from the hyperthermophilic archaeon, Pyrococcus furiosus (PfuPCNA) at 2.1 A resolution, and found that the toroidal ring-shaped structure, which consists of homotrimeric molecules, is highly conserved between the Eukarya and Archaea. This allowed us to examine its interaction with the loading factor at the atomic level. RESULTS: The replication factor C (RFC) is known as the loading factor of PCNA on to the DNA strand. P. furiosus RFC (PfuRFC) has a PCNA binding motif (PIP-box) at the C-terminus of the large subunit (RFCL). An 11 residue-peptide containing a PIP-box sequence of RFCL inhibited the PCNA-dependent primer extension ability of P. furiosus PolI in a concentration-dependent manner. To understand the molecular interaction mechanism of PCNA with PCNA binding proteins, we solved the crystal structure of PfuPCNA complexed with the PIP-box peptide. The interaction mode of the two molecules is remarkably similar to that of human PCNA and a peptide containing the PIP-box of p21(WAF1/CIP1). Moreover, the PIP-box binding may have some effect on the stability of the ring structure of PfuPCNA by some domain shift. CONCLUSIONS: Our structural analysis on PfuPCNA suggests that the interaction mode of the PIP-box with PCNA is generally conserved among the PCNA interacting proteins and that the functional meaning of the interaction via the PIP-box possibly depends on each protein. A movement of the C-terminal region of the PCNA monomer by PIP-box binding may cause the PCNA ring to be more rigid, suitable for its functions.

Amino Acid Sequence↗