A second proliferating cell nuclear antigen loader complex, Ctf18-replication factor C, stimulates DNA polymerase eta activity.
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, eta and lambda, in the most purified fraction, but experiments with purified recombinant proteins demonstrated that only polymerase (pol) eta was responsible for activity. Ctf18-RFC alone stimulated pol eta, and the addition of PCNA cooperatively increased stimulation. Furthermore, Ctf18-RFC interacted physically with pol eta, 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.
Shiomi
Yasushi
Y
Department of Biology, School of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Masutani
Chikahide
C
Hanaoka
Fumio
F
Kimura
Hiroshi
H
Tsurimoto
Toshiki
T
D000074183:ATPases Associated with Diverse Cellular Activities; D002352:Carrier Proteins / Q000378:metabolism*; D049109:Cell Proliferation; D002853:Chromatography, Liquid / Q000379:methods; D002875:Chromosomes / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism / Q000502:physiology*; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D051818:Replication Protein C / Q000378:metabolism*; D000099270:Y-Family DNA Polymerases
D000074183:ATPases Associated with Diverse Cellular Activities; D002352:Carrier Proteins; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D049452:Green Fluorescent Proteins; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D051818:Replication Protein C; C477290:CHTF18 protein, human; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases