Stable interaction between the human proliferating cell nuclear antigen loader complex Ctf18-replication factor C (RFC) and DNA polymerase {epsilon} is mediated by the cohesion-specific subunits, Ctf18, Dcc1, and Ctf8.
One of the proliferating cell nuclear antigen loader complexes, Ctf18-replication factor C (RFC), is involved in sister chromatid cohesion. To examine its relationship with factors involved in DNA replication, we performed a proteomics analysis of Ctf18-interacting proteins. We found that Ctf18 interacts with a replicative DNA polymerase, DNA polymerase ε (pol ε). Co-immunoprecipitation with recombinant Ctf18-RFC and pol ε demonstrated that their binding is direct and mediated by two distinct interactions, one weak and one stable. Three subunits that are specifically required for cohesion in yeast, Ctf18, Dcc1, and Ctf8, formed a trimeric complex (18-1-8) and together enabled stable binding with pol ε. The C-terminal 23-amino acid stretch of Ctf18 was necessary for the trimeric association of 18-1-8 and was required for the stable interaction. The weak interaction was observed with alternative loader complexes including Ctf18-RFC(5), which lacks Dcc1 and Ctf8, suggesting that the common loader structures, including the RFC small subunits (RFC2-5), are responsible for the weak interaction. The two interaction modes, mediated through distinguishable structures of Ctf18-RFC, both occurred through the N-terminal half of pol ε, which includes the catalytic domain. The addition of Ctf18-RFC or Ctf18-RFC(5) to the DNA synthesis reaction caused partial inhibition and stimulation, respectively. Thus, Ctf18-RFC has multiple interactions with pol ε that promote polymorphic modulation of DNA synthesis. We propose that their interaction alters the DNA synthesis mode to enable the replication fork to cooperate with the establishment of cohesion.
Murakami
Takeshi
T
From the Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
Takano
Ryuji
R
Takeo
Satoshi
S
Taniguchi
Rina
R
Ogawa
Kaori
K
Ohashi
Eiji
E
Tsurimoto
Toshiki
T
D000074183:ATPases Associated with Diverse Cellular Activities; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D004257:DNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism*; D051818:Replication Protein C / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
C477290:CHTF18 protein, human; C477291:CHTF8 protein, human; D002352:Carrier Proteins; C477292:DSCC1 protein, human; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; C497564:RFC5 protein, human; D004247:DNA; D004257:DNA Polymerase II; D000074183:ATPases Associated with Diverse Cellular Activities; D051818:Replication Protein C
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