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

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

doi:10.1007/s00792-002-0308-1

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

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

Molecular Biosciences

Research output: Contribution to journal › Article › peer-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 language	English
Pages (from-to)	169-175
Number of pages	7
Journal	Extremophiles
Volume	7
Issue number	3
DOIs	https://doi.org/10.1007/s00792-002-0308-1
Publication status	Published - Jun 1 2003

All Science Journal Classification (ASJC) codes

Microbiology
Molecular Medicine

Access to Document

10.1007/s00792-002-0308-1

Cite this

@article{f15aca4f809a4e31ae85b2231dc0464d,

title = "Mutational analysis of Pyrococcus furiosus replication factor C based on the three-dimensional structure",

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-{\AA} 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.",

author = "Sonoko Ishino and Takuji Oyama and Mihoko Yuasa and Kosuke Morikawa and Yoshizumi Ishino",

year = "2003",

month = jun,

day = "1",

doi = "10.1007/s00792-002-0308-1",

language = "English",

volume = "7",

pages = "169--175",

journal = "Extremophiles",

issn = "1431-0651",

publisher = "Springer Japan",

number = "3",

}

TY - JOUR

T1 - Mutational analysis of Pyrococcus furiosus replication factor C based on the three-dimensional structure

AU - Ishino, Sonoko

AU - Oyama, Takuji

AU - Yuasa, Mihoko

AU - Morikawa, Kosuke

AU - Ishino, Yoshizumi

PY - 2003/6/1

Y1 - 2003/6/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=1542649458&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=1542649458&partnerID=8YFLogxK

U2 - 10.1007/s00792-002-0308-1

DO - 10.1007/s00792-002-0308-1

M3 - Article

C2 - 12768447

AN - SCOPUS:1542649458

SN - 1431-0651

VL - 7

SP - 169

EP - 175

JO - Extremophiles

JF - Extremophiles

IS - 3

ER -

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

Abstract

All Science Journal Classification (ASJC) codes

Access to Document

Other files and links

Fingerprint

Cite this