Abstract
Background: DNA polymerase ɛ (Pol ɛ) 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 ɛ, we have been characterizing the interaction between Pol ɛ and DNA.
Results: Analysis of the four‐subunit Pol ɛ 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 ɛ does not require DNA ends or specific DNA sequences. Further analysis by competition experiments indicated that Pol ɛ 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 ɛ is inhibited by ssDNA but not by dsDNA. Furthermore, purification of the Pol ɛ auxiliary subunits Dpb3p and Dpb4p revealed that these proteins form a heterodimer and associate with dsDNA.
Conclusions: Pol ɛ 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 ɛ is inferred.