Abstract
DNA polymerase epsilon (pol epsilon) is a multiple subunit complex consisting of at least four proteins, including catalytic Pol2p, Dpb2p, Dpb3p, and Dpb4p. Pol epsilon has been shown to play essential roles in chromosomal DNA replication. Here, we report reconstitution of the yeast pol epsilon complex, which was expressed and purified from baculovirus-infected insect cells. During the purification, we were able to resolve the pol epsilon complex and truncated Pol2p (140 kDa), as was observed initially with the pol epsilon purified from yeast. Biochemical characterization of subunit stoichiometry, salt sensitivity, processivity, and stimulation by proliferating cell nuclear antigen indicates that the reconstituted pol epsilon is functionally identical to native pol epsilon purified from yeast and is therefore useful for biochemical characterization of the interactions of pol epsilon with other replication, recombination, and repair proteins. Identification and characterization of a proliferating cell nuclear antigen consensus interaction domain on Pol2p indicates that the motif is dispensable for DNA replication but is important for methyl methanesulfonate damage-induced DNA repair. Analysis of the putative zinc finger domain of Pol2p for zinc binding capacity demonstrates that it binds zinc. Mutations of the conserved cysteines in the putative zinc finger domain reduced zinc binding, indicating that cysteine ligands are directly involved in binding zinc.
Highlights
DNA polymerase ⑀ is a multiple subunit complex consisting of at least four proteins, including catalytic Pol2p, Dpb2p, Dpb3p, and Dpb4p
Biochemical characterization of subunit stoichiometry, salt sensitivity, processivity, and stimulation by proliferating cell nuclear antigen indicates that the reconstituted pol ⑀ is functionally identical to native pol ⑀ purified from yeast and is useful for biochemical characterization of the interactions of pol ⑀ with other replication, recombination, and repair proteins
Along with the previously validated vectors encoding the remaining subunits [8], we have devised a rapid purification of a highly active four-subunit holoenzyme. We show that this preparation is suitable for studying the interaction between pol ⑀ and other proteins by demonstrating that it is stimulated by the accessory proteins RF-C and PCNA, in just the same way as the enzyme from yeast (9 –11)
Summary
Despite these advances in studying pairwise interactions and even interactions between the two heterodimers, Pol2p/Dpb2p and Dpb3p/Dpb4p, we were not able to reconstitute a holoenzyme containing all four subunits with the same stoichiometry as the holoenzyme isolated from yeast Such reconstitution is critical for the step in our planned analysis, namely, to investigate how the holoenzyme interacts with its accessory factors, RF-C and PCNA in primer binding and processivity, as well as how it interacts at the mechanistic level with other proteins in replication, recombination, and repair. Along with the previously validated vectors encoding the remaining subunits [8], we have devised a rapid purification of a highly active four-subunit holoenzyme We show that this preparation is suitable for studying the interaction between pol ⑀ and other proteins by demonstrating that it is stimulated by the accessory proteins RF-C and PCNA, in just the same way as the enzyme from yeast (9 –11). Dimerization of Pol2p by Dpb2p [6, 14], is apparently not involved in zinc binding but is important for viability
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