Functional sites of human PCNA which interact with p21 (Cip1/Waf1), DNA polymerase δ and replication factor C

DNA replication efficiency is dictated by DNA polymerases (pol) and their associated proteins. The recent discovery of DNA polymerase Y family (DinB/UmuC/RAD30/REV1 superfamily) raises a question of whether the DNA polymerase activities are modified by accessory proteins such as proliferating cell nuclear antigen (PCNA). In fact, the activity of DNA pol IV (DinB) ofEscherichia coliis enhanced upon interaction with the β subunit, the processivity factor of DNA pol III. Here, we report the activity ofSsoDNA pol Y1 encoded by thedbhgene of the archaeonSulfolobus solfataricusis greatly enhanced by the presence of PCNA and replication factor C (RFC).Ssopol Y1per sewas a distributive enzyme but a substantial increase in the processivity was observed on poly(dA)-oligo(dT) in the presence of PCNA (039p or 048p) and RFC. The length of the synthesized DNA product reached at least 200 nucleotides.Ssopol Y1 displayed a higher affinity for DNA compared with pol IV ofE. coli, suggesting that the two DNA polymerases have distinct reason(s) to require the processivity factors for efficient DNA synthesis. The abilities of pol Y1 and pol IV to bypass DNA lesions and their sensitive sites to protease are also discussed.

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Faculty Opinions recommendation of Synthetic activity of Sso DNA polymerase Y1, an archaeal DinB-like DNA polymerase, is stimulated by processivity factors proliferating cell nuclear antigen and replication factor C.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1002546.28304 ◽

2001 ◽

Author(s):

Errol C Friedberg

Keyword(s):

Dna Polymerase ◽

Proliferating Cell Nuclear Antigen ◽

Nuclear Antigen ◽

Synthetic Activity ◽

Replication Factor C ◽

Replication Factor ◽

Factor C ◽

Proliferating Cell

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

Journal of Biological Chemistry ◽

10.1074/jbc.m610102200 ◽

2007 ◽

Vol 282 (29) ◽

pp. 20906-20914 ◽

Cited By ~ 21

Author(s):

Yasushi Shiomi ◽

Chikahide Masutani ◽

Fumio Hanaoka ◽

Hiroshi Kimura ◽

Toshiki Tsurimoto

Keyword(s):

Dna Polymerase ◽

Proliferating Cell Nuclear Antigen ◽

Nuclear Antigen ◽

Replication Factor C ◽

Replication Factor ◽

Factor C ◽

Proliferating Cell

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Replication Factor C Recruits DNA Polymerase δ to Sites of Nucleotide Excision Repair but Is Not Required for PCNA Recruitment

Molecular and Cellular Biology ◽

10.1128/mcb.00285-10 ◽

2010 ◽

Vol 30 (20) ◽

pp. 4828-4839 ◽

Cited By ~ 36

Author(s):

René M. Overmeer ◽

Audrey M. Gourdin ◽

Ambra Giglia-Mari ◽

Hanneke Kool ◽

Adriaan B. Houtsmuller ◽

...

Keyword(s):

Dna Synthesis ◽

Nucleotide Excision Repair ◽

Dna Polymerase ◽

Excision Repair ◽

Cell Protein ◽

Replication Factor C ◽

Replication Factor ◽

Nucleotide Excision ◽

Factor C ◽

Dna Repair Synthesis

ABSTRACT Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre- and postincision complexes. The postincision step of NER includes gap-filling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of the factors involved are not well understood. Using immunofluorescence, chromatin immunoprecipitation, and live-cell protein dynamic studies, we show that replication factor C (RFC) is implicated in postincision NER in mammalian cells. Small interfering RNA-mediated knockdown of RFC impairs upstream removal of UV lesions and abrogates the downstream recruitment of DNA polymerase delta. Unexpectedly, RFC appears dispensable for PCNA recruitment yet is required for the subsequent recruitment of DNA polymerases to PCNA, indicating that RFC is essential to stably load the polymerase clamp to start DNA repair synthesis at 3′ termini. The kinetic studies are consistent with a model in which RFC exchanges dynamically at sites of repair. However, its persistent localization at stalled NER complexes suggests that RFC remains targeted to the repair complex even after loading of PCNA. We speculate that RFC associates with the downstream 5′ phosphate after loading; such interaction would prevent possible signaling events initiated by the RFC-like Rad17 and may assist in unloading of PCNA.

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