A proteomics approach to identify proliferating cell nuclear antigen (PCNA)-binding proteins in human cell lysates. Identification of the human CHL12/RFCs2-5 complex as a novel PCNA-binding protein

Proliferating cell nuclear antigen (PCNA), a eukaryotic DNA replication factor, functions not only as a processivity factor for DNA polymerase delta but also as a binding partner for multiple other factors. To understand its broad significance, we have carried out systematic studies of PCNA-binding...

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Bibliographic Details
Published in:The Journal of biological chemistry 2002-10, Vol.277 (43), p.40362-40367
Main Authors: Ohta, Satoshi, Shiomi, Yasushi, Sugimoto, Katsunori, Obuse, Chikashi, Tsurimoto, Toshiki
Format: Article
Language:English
Subjects:
Base Sequence
Chromatography, Affinity
Chromatography, Liquid
DNA Primers
Electrophoresis, Polyacrylamide Gel
Humans
Mass Spectrometry
Proliferating Cell Nuclear Antigen - metabolism
Protein Binding
Proteomics
Replication Protein C
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Summary:Proliferating cell nuclear antigen (PCNA), a eukaryotic DNA replication factor, functions not only as a processivity factor for DNA polymerase delta but also as a binding partner for multiple other factors. To understand its broad significance, we have carried out systematic studies of PCNA-binding proteins by a combination of affinity chromatography and mass spectrometric analyses. We detected more than 20 specific protein bands of various intensities in fractions bound to PCNA-fixed resin from human cell lysates and determined their peptide sequences by liquid chromatography and tandem mass spectrometry. A search with human protein data bases identified 12 reported PCNA-binding proteins from both cytoplasmic (S100 lysate) and nuclear extracts with substantial significance and four more solely from the nuclear preparation. CHL12, a factor involved in checkpoint response and chromosome cohesion, was a novel example found in both lysates. Further studies with recombinant proteins demonstrated that CHL12 and small subunits of replication factor C form a complex that interacts with PCNA.
ISSN:0021-9258
DOI:10.1074/jbc.M206194200