Characterization of Human DNA Polymerase Delta and Its Subassemblies Reconstituted by Expression in the Multibac System (original) (raw)

Abstract

Mammalian DNA polymerase d (Pol d), a four-subunit enzyme, plays a crucial and versatile role in DNA replication and DNA repair processes. We have reconstituted human Pol d complexes in insect cells infected with a single baculovirus into which one or more subunits were assembled. This system allowed for the efficient expression of the tetrameric Pol d holoenzyme, the p125/p50 core dimer, the core+p68 trimer and the core+p12 trimer, as well as the p125 catalytic subunit. These were isolated in milligram amounts with reproducible purity and specific activities by a highly standardized protocol. We have systematically compared their activities in order to gain insights into the roles of the p12 and p68 subunits, as well as their responses to PCNA. The relative specific activities (apparent k cat) of the Pol d holoenzyme, core+p68, core+p12 and p125/ p50 core were 100, 109, 40, and 29. The corresponding apparent K d 's for PCNA were 7.1, 8.7, 9.3 and 73 nM. Our results support the hypothesis that Pol d interacts with PCNA through multiple interactions, and that there may be a redundancy in binding interactions that may permit Pol d to adopt flexible configurations with PCNA. The abilities of the Pol d complexes to fully extend singly primed M13 DNA were examined. All the subassemblies except the core+p68 were defective in their abilities to completely extend the primer, showing that the p68 subunit has an important function in synthesis of long stretches of DNA in this assay. The core+p68 trimer could be reconstituted by addition of p12.

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