title: Double-stranded DNA binding properties of Saccharomyces cerevisiae DNA polymerase epsilon and of the Dpb3p-Dpb4p subassembly.
abstract: BACKGROUND
  DNA polymerase epsilon (Pol epsilon) of Saccharomyces cerevisiae participates in many aspects of DNA replication, as well as in DNA repair. In order to clarify molecular mechanisms employed in the multiple tasks of Pol epsilon, we have been characterizing the interaction between Pol epsilon and DNA.
  
  
  RESULTS
  Analysis of the four-subunit Pol epsilon complex by gel mobility shift assay revealed that the complex binds not only to single-stranded (ss) DNA but also equally well to double-stranded (ds) DNA. A truncated polypeptide consisting of the N-terminal domain of Pol2p catalytic subunit binds to ssDNA but not to dsDNA, indicating that the Pol2p C-terminal domain and/or the auxiliary subunits are involved in the dsDNA-binding. The dsDNA-binding by Pol epsilon does not require DNA ends or specific DNA sequences. Further analysis by competition experiments indicated that Pol epsilon contains at least two distinct DNA-binding sites, one of which binds exclusively to ssDNA and the other to dsDNA. The dsDNA-binding site, however, is suggested to also bind ssDNA. The DNA polymerase activity of Pol epsilon is inhibited by ssDNA but not by dsDNA. Furthermore, purification of the Pol epsilon auxiliary subunits Dpb3p and Dpb4p revealed that these proteins form a heterodimer and associate with dsDNA.
  
  
  CONCLUSIONS
  Pol epsilon has multiple sites at which it interacts with DNA. One of these sites has a strong affinity for dsDNA, a feature that is not generally associated with DNA polymerases. Involvement of the Dpb3p-Dpb4p complex in the dsDNA-binding of Pol epsilon is inferred.
authors: Department of Molecular Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan.
authors: Toshiaki
authors: T
authors: Tsubota
authors: Satoko
authors: S
authors: Maki
authors: Hajime
authors: H
authors: Kubota
authors: Akio
authors: A
authors: Sugino
authors: Hisaji
authors: H
authors: Maki
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D004257:DNA Polymerase II / Q000378:metabolism*; D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
chemical_list: D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D004257:DNA Polymerase II