TY - JOUR
T1 - A second proliferating cell nuclear antigen loader complex, Ctf18-replication factor C, stimulates DNA polymerase η activity
AU - Shiomi, Yasushi
AU - Masutani, Chikahide
AU - Hanaoka, Fumio
AU - Kimura, Hiroshi
AU - Tsurimoto, Toshiki
PY - 2007/7/20
Y1 - 2007/7/20
N2 - Replication factor C (RFC) loads the clamp protein PCNA ontoDNAstructures. 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, η and λ, in the most purified fraction, but experiments with purified recombinant proteins demonstrated that only polymerase (pol) η was responsible for activity. Ctf18-RFC alone stimulated pol η, and the addition of PCNA cooperatively increased stimulation. Furthermore, Ctf18-RFC interacted physically with pol η, 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.
AB - Replication factor C (RFC) loads the clamp protein PCNA ontoDNAstructures. 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, η and λ, in the most purified fraction, but experiments with purified recombinant proteins demonstrated that only polymerase (pol) η was responsible for activity. Ctf18-RFC alone stimulated pol η, and the addition of PCNA cooperatively increased stimulation. Furthermore, Ctf18-RFC interacted physically with pol η, 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.
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U2 - 10.1074/jbc.M610102200
DO - 10.1074/jbc.M610102200
M3 - Article
C2 - 17545166
AN - SCOPUS:34547096484
SN - 0021-9258
VL - 282
SP - 20906
EP - 20914
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 29
ER -