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Molecular and Cellular Biology, November 1999, p. 7428-7435, Vol. 19, No. 11
Department of Viral Oncology, Institute for
Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan.
Received 26 May 1999/Returned for modification 7 July 1999/Accepted 9 August 1999
Evidence for transcription factor involvement in the initiation of
DNA replication at certain replication origins in Saccharomyces cerevisiae mainly comes from an indirect assay which measures the
mitotic stability of plasmids containing an autonomously replicating sequence (ARS), a selectable marker gene, and a centromere. In order to
eliminate the effect of transcription factor binding to the selectable
marker gene or centromere in such assays, we have adapted the
DpnI assay to directly measure ARS replication activity in
vivo by using ARS plasmids devoid of extraneous transcription elements.
Using this assay, we found that the B3 element of ARS1, which serves as
a binding site for the transcription factor Abf1p, does not stimulate
ARS activity on plasmids lacking a centromere and a selectable marker
gene. We also found with such plasmids that exogenous expression of the
strong transcriptional activators Gal4 and Gal4-VP16 inhibited the
replication activity of ARS1 when B3 was replaced by the Gal4 binding
site, although these activators had previously been shown to stimulate
replication activity in the stability assay. Moreover, a chromosomally
inactive ARS, ARS301, which was active by itself on a plasmid, was
inactivated by placing an Abf1p binding site in its vicinity. These
results indicate that the sequences surrounding the ARS as well as
properties of the ARS element itself determine its response to
transcription factors.
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Context-Dependent Modulation of Replication
Activity of Saccharomyces cerevisiae Autonomously
Replicating Sequences by Transcription Factors
*
Corresponding author. Mailing address: Department of
Viral Oncology, Institute for Virus Research, Kyoto University,
Shogoinkawahara-machi, Sakyo-ku, Kyoto 606-8507, Japan. Phone:
81-75-751-4030. Fax: 81-75-752-3232. E-mail:
yota{at}virus.kyoto-u.ac.jp.
Molecular and Cellular Biology, November 1999, p. 7428-7435, Vol. 19, No. 11
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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