Abstract
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.
Highlights
At least three essential DNA polymerases, ␣, ⑀, and ␦, play critical roles in DNA replication [1,2,3]
Structure of DNA Polymerase ␦—The results presented here indicate that the baculovirus-expressed 4S S. pombe pol ␦ complex is monomeric
We reported previously that these complexes were dimeric based on their elution profile from a sizing column. We realize that this difference is due to the elongated shape of the 4S complex caused by the highly elongated structure of the Cdc27 subunit
Summary
At least three essential DNA polymerases, ␣, ⑀, and ␦, play critical roles in DNA replication [1,2,3]. SV40 viral DNA replication, carried out with highly purified proteins, has defined the role of DNA polymerase ␣ (pol ␣) in the initiation of both leading and lagging strands. ␣-associated primase subunits synthesize small oligoribonucleotides (10 –15 nucleotides long) that are elongated for a short distance (ϳ35 nucleotides) by the large catalytic subunit of pol ␣ [1, 7, 8] These short RNA-DNA segments are elongated by pol ␦ or pol ⑀ via a series of reactions in which the pol ␣-primase complex is displaced by RFC and PCNA [9, 10]. The heterotrimeric S. cerevisiae pol ␦ is highly processive, whereas the heterodimer, Pol3p-Pol31p, is less processive, in keeping with observations made in S. pombe
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