title: Human CTF18-RFC clamp-loader complexed with non-synthesising DNA polymerase ε efficiently loads the PCNA sliding clamp.
abstract: The alternative proliferating-cell nuclear antigen (PCNA)-loader CTF18-RFC forms a stable complex with DNA polymerase ε (Polε). We observed that, under near-physiological conditions, CTF18-RFC alone loaded PCNA inefficiently, but loaded it efficiently when complexed with Polε. During efficient PCNA loading, CTF18-RFC and Polε assembled at a 3΄ primer-template junction cooperatively, and directed PCNA to the loading site. Site-specific photo-crosslinking of directly interacting proteins at the primer-template junction showed similar cooperative binding, in which the catalytic N-terminal portion of Polε acted as the major docking protein. In the PCNA-loading intermediate with ATPγS, binding of CTF18 to the DNA structures increased, suggesting transient access of CTF18-RFC to the primer terminus. Polε placed in DNA synthesis mode using a substrate DNA with a deoxidised 3΄ primer end did not stimulate PCNA loading, suggesting that DNA synthesis and PCNA loading are mutually exclusive at the 3΄ primer-template junction. Furthermore, PCNA and CTF18-RFC-Polε complex engaged in stable trimeric assembly on the template DNA and synthesised DNA efficiently. Thus, CTF18-RFC appears to be involved in leading-strand DNA synthesis through its interaction with Polε, and can load PCNA onto DNA when Polε is not in DNA synthesis mode to restore DNA synthesis.
authors.affiliation: Department of Biology, Faculty of Science, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Fujisawa
authors.affiliation: Department of Biology, Faculty of Science, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Ohashi
authors.affiliation: Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, Minami-Osawa 1-1, Hachioji-shi, Tokyo 192-0397, Japan.
authors.forename: Kouji
authors.initials: K
authors.lastname: Hirota
authors.affiliation: Department of Biology, Faculty of Science, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Toshiki
authors.initials: T
authors.lastname: Tsurimoto
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004257:DNA Polymerase II / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D000075223:Poly-ADP-Ribose Binding Proteins; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D051818:Replication Protein C / Q000235:genetics* / Q000378:metabolism
chemical_list: C477290:CHTF18 protein, human; D002352:Carrier Proteins; D017931:DNA Primers; D009687:Nuclear Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; C081632:RFC1 protein, human; D011994:Recombinant Proteins; D004247:DNA; D004257:DNA Polymerase II; C119525:POLE protein, human; D000074183:ATPases Associated with Diverse Cellular Activities; D051818:Replication Protein C