Identification of a novel protein, PDIP38, that interacts with the p50 subunit of DNA polymerase delta and proliferating cell nuclear antigen.
The yeast two-hybrid screening method was used to identify novel proteins that associate with human DNA polymerase delta (pol delta). Two baits were used in this study. These were the large (p125) and small (p50) subunits of the core pol delta heterodimer. p50 was the only positive isolated with p125 as the bait. Two novel protein partners, named PDIP38 and PDIP46, were identified from the p50 screen. In this study, the interaction of PDIP38 with pol delta was further characterized. PDIP38 encodes a protein of 368 amino acids whose C terminus is conserved with the bacterial APAG protein and with the F box A protein. It was found that PDIP38 also interacts with proliferating cell nuclear antigen (PCNA). The ability of PDIP38 to interact with both the p50 subunit of pol delta and with PCNA was confirmed by pull-down assays using glutathione S-transferase (GST)-PDIP38 fusion proteins. The PCNA-PDIP38 interaction was also demonstrated by PCNA overlay experiments. The association of PDIP38 with pol delta was shown to occur in calf thymus tissue and mammalian cell extracts by GST-PDIP38 pull-down and coimmunoprecipitation experiments. PDIP38 was associated with pol delta isolated by immunoaffinity chromatography. The association of PDIP38 with pol delta could also be demonstrated by native gel electrophoresis.
Liu
Li
L
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla 10595, USA.
Rodriguez-Belmonte
Esther M
EM
Mazloum
Nayef
N
Xie
Bin
B
Lee
Marietta Y W T
MY
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002846:Chromatography, Affinity; D004258:DNA Polymerase III / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011233:Precipitin Tests; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D021122:Protein Subunits; D020798:Two-Hybrid System Techniques
D009687:Nuclear Proteins; C473591:PDIP38 protein, mouse; C473592:POLDIP2 protein, human; D018809:Proliferating Cell Nuclear Antigen; D021122:Protein Subunits; D004258:DNA Polymerase III