DNA polymerase II (epsilon) of Saccharomyces cerevisiae dissociates from the DNA template by sensing single-stranded DNA.
Two forms of DNA polymerase II (epsilon) of Saccharomyces cerevisiae, Pol II* and Pol II, were purified to near homogeneity from yeast cells. Pol II* is a four-subunit complex containing a 256-kDa catalytic polypeptide, whereas Pol II consists solely of a 145-kDa polypeptide derived from the N-terminal half of the 256-kDa polypeptide of Pol II*. We show that Pol II* and Pol II are indistinguishable with respect to the processivity and rate of DNA-chain elongation. The equilibrium dissociation constants of the complexes of Pol II* and Pol II with the DNA template showed that the stability of these complexes is almost the same. However, when the rates of dissociation of the Pol II* and Pol II from the DNA template were measured using single-stranded DNA as a trap for the dissociated polymerase, Pol II* dissociated 75-fold faster than Pol II. Furthermore, the rate of dissociation of Pol II* from the DNA template became faster as the concentration of the single-stranded DNA was increased. These results indicate that the rapid dissociation of Pol II* from the DNA template is actively promoted by single-stranded DNA. The dissociation of Pol II from the DNA template was also shown to be promoted by single-stranded DNA, although at a much slower rate. These results suggest that the site for sensing single-stranded DNA resides within the 145-kDa N-terminal portion of the catalytic subunit and that the efficiency for sensing single-stranded DNA by this site is positively modulated by either the C-terminal half of the catalytic subunit and/or the other subunits.
Department of Biochemistry and Molecular Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
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Maki
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Hashimoto
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Ohara
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Sugino
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D004257:DNA Polymerase II / Q000378:metabolism*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013698:Templates, Genetic
D004277:DNA, Single-Stranded; D004257:DNA Polymerase II