Double-stranded DNA binding, an unusual property of DNA polymerase epsilon, promotes epigenetic silencing in Saccharomyces cerevisiae.
We have previously shown that DNA polymerase epsilon (Pol epsilon)of Saccharomyces cerevisiae binds stably to double-stranded DNA (dsDNA), a property not generally associated with DNA polymerases. Here, by reconstituting Pol epsilon activity from Pol2p-Dpb2p and Dpb3p-Dpb4p, its two component subassemblies, we report that Dpb3p-Dpb4p, a heterodimer of histone-fold motif-containing subunits, is responsible for the dsDNA binding. Substitution of specific lysine residues in Dpb3p, highlighted by homology modeling of Dpb3p-Dpb4p based on the structure of the histone H2A-H2B dimer, indicated that they play roles in binding of dsDNA by Dpb3p-Dpb4p, in a manner similar to the histone-DNA interaction. The lysine-substituted dpb3 mutants also displayed reduced telomeric silencing, whose degree paralleled that of the dsDNA-binding activity of Pol epsilon in the corresponding dpb3 mutants. Furthermore, additional amino acid substitutions to lysines in Dpb4p, to compensate for the loss of positive charges in the Dpb3p mutants, resulted in simultaneous restoration of dsDNA-binding activity by Pol epsilon and telomeric silencing. We conclude that the dsDNA-binding property of Pol epsilon is required for epigenetic silencing at telomeres.
Tsubota
Toshiaki
T
Department of Molecular Biology, Graduate School of Biological Sciences and Graduate School of Information Science, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan.
Tajima
Rie
R
Ode
Kunitomo
K
Kubota
Hajime
H
Fukuhara
Naoshi
N
Kawabata
Takeshi
T
Maki
Satoko
S
Maki
Hisaji
H
D019943:Amino Acid Substitution; D004257:DNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D044127:Epigenesis, Genetic; D020868:Gene Silencing; D005800:Genes, Fungal; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D004271:DNA, Fungal; D004257:DNA Polymerase II