Structure and activity associated with multiple forms of Schizosaccharomyces pombe DNA polymerase delta.
DNA polymerase delta (Pol delta) isolated from Schizosaccharomyces pombe (sp) consists of at least four subunits, Pol3, Cdc1, Cdc27, and Cdm1. We have reconstituted the four-subunit complex by simultaneously expressing these polypeptides in baculovirus-infected insect cells. The properties of the purified cloned spPol delta were identical to the native spPol delta isolated from S. pombe cells. In addition, we also isolated a three-subunit complex containing Pol3, Cdc1, and Cdm1. Both three- and four-subunit complexes required replication factor C and proliferating cell nuclear antigen for DNA replication. However, in the presence of low levels of polymerase complexes, the three-subunit complex was less efficient than the four-subunit complex in supporting DNA replication. The inefficient synthesis of DNA by the three-subunit complex can be remedied by the addition of Cdc27, the subunit missing in the three-subunit complex. Gel filtration analysis demonstrated that the three-subunit complex is a monomer of the heterotrimer (Pol3, Cdc1, and Cdm1) and that the four-subunit complex is a dimer of the heterotetramer (Pol3, Cdc1, Cdc27, and Cdm1), similar to the structure of native spPol delta. We have further shown that Cdc1 and Cdc27 interact to form a heterodimeric complex. Gel filtration studies indicate that the structure of this complex is dimeric. These observations suggest that the Cdc27 subunit may play an important role contributing to the dimerization of Pol delta.
Zuo
S
S
Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Bermudez
V
V
Zhang
G
G
Kelman
Z
Z
Hurwitz
J
J
D018797:Cell Cycle Proteins / Q000378:metabolism; D004258:DNA Polymerase III / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins; D013329:Structure-Activity Relationship
D018797:Cell Cycle Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; C513024:cdc27 protein, S pombe; D004258:DNA Polymerase III