RESUMO
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.
Assuntos
Proteínas de Transporte/metabolismo , DNA Polimerase II/metabolismo , Replicação do DNA/fisiologia , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína de Replicação C/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Sítios de Ligação , Proteínas de Transporte/genética , DNA/biossíntese , DNA/genética , DNA Polimerase II/genética , Células HeLa , Humanos , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Antígeno Nuclear de Célula em Proliferação/genética , Proteína de Replicação C/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Calix[4]arene-based triple helicates take up an achiral part of chiral guests; the chiral center outside the cavity causes the asymmetric induction of their supramolecular helicity.
