Stable Interaction between the Human Proliferating Cell Nuclear Antigen Loader Complex Ctf18-Replication Factor C (RFC) and DNA Polymerase ϵ Is Mediated by the Cohesion-specific Subunits, Ctf18, Dcc1, and Ctf8

Abstract

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 ⑀ (pol ⑀). Co-immunoprecipitation with recombinant Ctf18-RFC and pol ⑀ 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 ⑀. 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 ⑀, 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 ⑀ 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.The eukaryotic loader complex replication factor C (RFC) 2 consists of one large and four small subunits (RFC1 and RFC2-5, respectively) and loads the clamp complex proliferating cell nuclear antigen (PCNA) onto a primer/template DNA through its ATPase activity. PCNA is a homotrimeric complex functioning as the processivity factor for replicative DNA polymerase ␦ (pol ␦). Three RFC1 paralogues, Ctf18, Rad17, and Elg1, exist in eukaryotes and function as the largest subunits of three alternative loader complexes, Ctf18-RFC, Rad17-RFC, and Elg1-RFC, respectively, in association with RFC2-5 (for review, see Refs. 1, 2). These loader complexes target PCNA or the alternative clamp, the Rad9-Hus1-Rad1 (9-1-1) complex, and contribute to various reactions tightly related to DNA replication, such as chromosome cohesion, DNA damage responses, and maintenance of genome stability (3, 4).Ctf18-RFC forms a heteroheptameric complex combining the pentameric core loader complex, Ctf18 and RFC2-5 (hereafter referred to as Ctf18-RFC(5)), and two additional cohesionspecific factors, Dcc1 and Ctf8 (5, 6), whose yeast orthologues are required for faithful sister chromatid cohesion (7-9). Ctf18-RFC loads functional PCNA onto DNA and stimulates pol ␦ (6, 10). Its PCNA-unloading activity depends on replication protein A concentration (11), but its functional significance in vivo has not been elucidated. We hav...

The essential kinase ATR: ensuring faithful duplication of a challenging genome

“…The 9-1-1 clamp is structurally similar to the proliferating cell nuclear antigen (PCNA) sliding clamp and is loaded onto the DNA by the RAD17–replication factor C subunits 2-5 (RFC2-5) clamp loader 30–32 . An alternative clamp loader, CTF18–RFC2-5, interacts with DNA polymerase epsilon (Pol ε) 33 , which is the enzyme that synthesizes the leading strand at the replication fork. In budding yeast, this interaction is a key step in the activation of the downstream replication checkpoint response 34,35 .…”

Human CTF18-RFC clamp-loader complexed with non-synthesising DNA polymerase ε efficiently loads the PCNA sliding clamp

“…RFC, CTF18-RFC, CTF18-RFC(5), PCNA, RPA, the four-subunit complex of DNA polymerase δ consisting of p125, p66, His-p50 and p12 and the DNA polymerase ε complex consisting of His-p261, FLAG-p59, p17 and p12 (hereafter referred to as Polδ and Polε, respectively, unless noted otherwise) were purified as described previously (27,30,31). …”

Phosphoinositide 3-kinase beta controls replication factor C assembly and function

“…Fractions (0.5 ml) were collected from the top and proteins analysed by WB. Standard sedimentation markers were loaded in parallel glycerol gradients to determine sedimentation position (25). …”