The human homologue of fission Yeast cdc27, p66, is a component of active human DNA polymerase delta.
An essential eukaryotic DNA polymerase, DNA polymerase delta (pol delta), synthesizes DNA processively in the presence of proliferating cell nuclear antigen (PCNA). Recently, a 66 kDa polypeptide (p66) that displays significant homology within its PCNA binding domain to that of fission yeast cdc27 was identified as a component of mouse and calf thymus pol delta. Our studies show that p66 interacts tightly with other subunits of pol delta during size fractionation of human cell extracts, and co-immunoprecipitates with these subunits along with PCNA-dependent polymerase activity. Active human pol delta could be reconstituted by co-expressing p125, p50, and p66 recombinant baculoviruses, but not by co-expressing p125 and p50 alone. Interaction studies demonstrated that p66 stabilizes the association between p125 and p50. Pull-down assays with PCNA-linked beads demonstrated that p66 increases the overall affinity of pol delta for PCNA. These results indicate that p66 is a functionally important subunit of human pol delta that stabilizes the pol delta complex and increases the affinity of pol delta for PCNA.
Shikata
K
K
Nara Institute of Science and Technology, Takayama, Ikoma, Nara 630-0101, Japan.
Ohta
S
S
Yamada
K
K
Obuse
C
C
Yoshikawa
H
H
Tsurimoto
T
T
D000818:Animals; D064191:Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome; D002417:Cattle; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability / Q000502:physiology; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry* / Q000378:metabolism; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000737:chemistry / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins; D017385:Sequence Homology
D064191:Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome; C577366:CDC27 protein, human; D018797:Cell Cycle Proteins; D010455:Peptides; D018809:Proliferating Cell Nuclear Antigen; D021122:Protein Subunits; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; C513024:cdc27 protein, S pombe; C435720:POLD3 protein, human; D004258:DNA Polymerase III