The influence of the Cdc27 subunit on the properties of the Schizosaccharomyces pombe DNA polymerase delta.
Schizosaccharomyces pombe DNA polymerase (pol) delta contains four subunits, pol 3, Cdc1, Cdc27, and Cdm1. In this report, we examined the role of Cdc27 on the structure and activity of pol delta. We show that the four-subunit complex is monomeric in structure, in contrast to the previous report that it was a dimer (Zuo, S., Bermudez, V., Zhang, G., Kelman, Z., and Hurwitz, J. (2000) J. Biol. Chem. 275, 5153-5162). This discrepancy between the earlier and recent observations was traced to the marked asymmetric shape of Cdc27. Cdc27 contains two critical domains that govern its role in activating pol delta. The N-terminal region (amino acids (aa) 1-160) binds to Cdc1 and its extreme C-terminal end (aa 362-369) interacts with proliferating cell nuclear antigen (PCNA). Mutants of S. pombe pol delta, containing truncated Cdc27 derivatives deficient in binding to PCNA, supported DNA replication less processively than the wild-type complex. Fusion of a minimal PCNA-binding motif (aa 352-372) to C-terminally truncated Cdc27 derivatives restored processive DNA synthesis in vitro. In vivo, the introduction of these fused Cdc27 derivatives into cdc27Delta cells conferred viability. These data support the model in which Cdc27 plays an essential role in DNA replication by recruiting PCNA to the pol delta holoenzyme.
Bermudez
Vladimir P
VP
Memorial Sloan-Kettering Cancer Center, Program of Molecular Biology, New York, New York 10021, USA.
MacNeill
Stuart A
SA
Tappin
Inger
I
Hurwitz
Jerard
J
D020816:Amino Acid Motifs; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D004258:DNA Polymerase III / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005816:Genetic Complementation Test; D008956:Models, Chemical; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins
D018797:Cell Cycle Proteins; D018809:Proliferating Cell Nuclear Antigen; D029702:Schizosaccharomyces pombe Proteins; C513024:cdc27 protein, S pombe; D004247:DNA; D004258:DNA Polymerase III