A Second Proliferating Cell Nuclear Antigen Loader Complex, Ctf18-Replication Factor C, Stimulates DNA Polymerase η Activity

Abstract

Replication factor C (RFC) loads the clamp protein PCNA onto DNA structures. Ctf18-RFC, which consists of the chromosome cohesion factors Ctf18, Dcc1, and Ctf8 and four small RFC subunits, functions as a second proliferating cell nuclear antigen (PCNA) loader. To identify potential targets of Ctf18-RFC, human cell extracts were assayed for DNA polymerase activity specifically stimulated by Ctf18-RFC in conjunction with PCNA. After several chromatography steps, an activity stimulated by Ctf18-RFC but not by RFC was identified. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis revealed the presence of two DNA polymerases, and , in the most purified fraction, but experiments with purified recombinant proteins demonstrated that only polymerase (pol) was responsible for activity. Ctf18-RFC alone stimulated pol , and the addition of PCNA cooperatively increased stimulation. Furthermore, Ctf18-RFC interacted physically with pol , as indicated by co-precipitation in human cells. We propose that this novel loader-DNA polymerase interaction allows DNA replication forks to overcome interference by various template structures, including damaged DNA and DNA-protein complexes that maintain chromosome cohesion.Clamp and clamp loader proteins recruit target proteins to DNA structures, such as replication forks and repair intermediates. A representative eukaryotic clamp, PCNA, 2 is loaded onto replicating DNA ends by a loader complex, RFC, and functions as a processivity factor for the replicative DNA polymerases ␦ and ⑀ (1). RFC is a heteropentameric protein complex composed of a large subunit (RFC1) and four small subunits (RFC2 to -5), all of which belong to the AAAϩ ATPase family (2, 3). Structural and biochemical studies have demonstrated that an ATP-dependent conformational change in RFC promotes loading of PCNA onto template DNA (4 -8). Three other clamp loader complexes that are involved in checkpoint responses (Rad17-RFC), sister chromatid cohesion (Ctf18/ Chl12-RFC), and maintenance of genome stability (Elg1-RFC) have been identified in eukaryotes (9, 10). Ctf18/Chl12, hereafter referred to as Ctf18, is required for precise chromosome transmission in yeast and is highly conserved in eukaryotes (11,12). Unlike other loader complexes, Ctf18-RFC also associates with the chromosome cohesion factors Dcc1 and Ctf8 to form a heteroheptameric complex, although functions for the additional subunits have not been identified (13) 3 ; we refer to the heptameric and pentameric complexes as Ctf18-RFC and Ctf18-RFC(5s), respectively. Ctf18-RFC loads functional PCNA onto the 3Ј end of a primer-template DNA duplex, and like RFC, it stimulates pol ␦ activity in vitro. Nonetheless, the functions of these two PCNA loaders are clearly distinguishable. For example, PCNA loaded by Ctf18-RFC can fully support DNA synthesis by pol ␦ on a primed M13mp18 singlestranded DNA (M13) DNA template, but Ctf18-RFC cannot substitute for RFC in promoting SV40 DNA replication in vitro with crude human protein fractions (14). These results ...

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

“…DNA Polymerase Assay-DNA synthesis was assayed as described previously (12) by measuring [ 32 P]TMP counts adsorbed to DE81 paper after incubation of the reaction mixture at 37°C for 30 min.…”

DNA methylation and chromatin dynamics during postnatal cardiomyocyte maturation

“…In addition, Chtf18 has been previously shown to drive cell cycle by forming a complex with the replication factor C to load proliferating cell nuclear antigen (PCNA) to DNA. DNA polymerase η will then be activated following the loading of PCNA to DNA to initiate DNA synthesis (Shiomi et al, 2007). Notably, Chtf18 is the only conserved DNA methylation target gene that was identified in this Chapter, supporting a previous study that has demonstrated its conservation for DNA replication between yeast, flies and mammals (Berkowitz et al, 2008).…”

“…Ube2c have been previously reported as cell cycle regulators (Shiomi et al, 2007, Gruneberg et al, 2006Liu et al, 2003, van Ree et al, 2010. Specifically, Cit can regulate cell cycle through forming a complex with Klf14 at central spindle and midbody during G2/M stage transition (Gruneberg et al, 2006;Liu et al, 2003).…”

Differential microRNA expression between bone marrow side population cells and hepatocytes in adult mice

“…The receipt of differentiation signals by HSCEs may cause a decrease in miR-127 levels, releasing them from a quiescent state to actively proliferate and differentiate. Moreover, a database search (miRBase; http://microrna.sanger.ac.uk/) also suggested the possibility that miR-207 might targeting Ctf18-replication factor C, which regulates the cell cycle (48). Nevertheless, miR-127 and -207 have many other candidate targets besides BCL6 and Ctf18, and they may modulate another pathway and/or another target in both SP cells and HSCEs.…”