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 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.
Ohta
Satoshi
S
Nara Institute of Science and Technology, Takayama, Ikoma, Nara 630-0101, Japan.
Shiomi
Yasushi
Y
Sugimoto
Katsunori
K
Obuse
Chikashi
C
Tsurimoto
Toshiki
T
D001483:Base Sequence; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D013058:Mass Spectrometry; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011485:Protein Binding; D040901:Proteomics; D051818:Replication Protein C
D017931:DNA Primers; D018809:Proliferating Cell Nuclear Antigen; C497561:RFC2 protein, human; C497562:RFC3 protein, human; C497563:RFC4 protein, human; C497564:RFC5 protein, human; D051818:Replication Protein C