Direct interaction of proliferating cell nuclear antigen with the small subunit of DNA polymerase delta.
The interaction between proliferating cell nuclear antigen (PCNA) and DNA polymerase delta is essential for processive DNA synthesis during DNA replication/repair; however, the identity of the subunit of DNA polymerase delta that directly interacts with PCNA has not been resolved until now. In the present study we have used reciprocal co-immunoprecipitation experiments to determine which of the two subunits of core DNA polymerase delta, the 125-kDa catalytic subunit or the 50-kDa small subunit, directly interacts with PCNA. We found that PCNA co-immunoprecipitated with human p50, as well as calf thymus DNA polymerase delta heterodimer, but not with p125 alone, suggesting that PCNA directly interacts with p50 but not with p125. A PCNA-binding motif, similar to the sliding clamp-binding motif of bacteriophage RB69 DNA polymerase, was identified in the N terminus of p50. A 22-amino acid oligopeptide containing this sequence (MRPFL) was shown to bind PCNA by far Western analysis and to compete with p50 for binding to PCNA in co-immunoprecipitation experiments. The binding of p50 to PCNA was inhibited by p21, suggesting that the two proteins compete for the same binding site on PCNA. These results establish that the interaction of PCNA with DNA polymerase delta is mediated through the small subunit of the enzyme.
Lu
Xiaoqing
X
Departments of Medicine and Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101, USA.
Tan
Cheng-Keat
CK
Zhou
Jin-Qiu
JQ
You
Min
M
Carastro
L Michael
LM
Downey
Kathleen M
KM
So
Antero G
AG
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D004258:DNA Polymerase III / Q000378:metabolism*; D004261:DNA Replication; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D013950:Thymus Gland / Q000201:enzymology; D014162:Transfection
D010446:Peptide Fragments; D018809:Proliferating Cell Nuclear Antigen; D021122:Protein Subunits; D011994:Recombinant Proteins; D004258:DNA Polymerase III