Nucleic Acids Research, Vol 25, Issue 24 5057-5064, Copyright © 1997 by Oxford University Press
S Raychaudhuri, R Byers, T Upton and S Eisenberg
Yeast replication origins have a modular arrangement of essential DNA
sequences containing the ARS consensus sequence (ACS) flanked by auxiliary
DNA elements which stimulate origin function. One of the auxiliary elements
identified at several origins is a DNA replication enhancer that binds the
Abf1p protein. We have isolated an ARS sequence from Saccharomyces
cerevisiae based on its ability to bind Abf1p. Here we present a detailed
molecular dissection of this ARS, designated ARS 1501, and we demonstrate
that it functions as a genomic replication origin on chromosome XV .
Mutagenesis of the Abf1p DNA-binding sites revealed that these sequences
did not contribute significantly to ARS function. Instead, a new DNA
element important for replication, designated REN1501, has been located 5'
to the T-rich strand of the ACS. We show that REN1501 functions in either
orientation and at variable distances from the ACS, defining this element
as a DNA replication enhancer. Most significantly, point mutations within
this element decreased the stability of plasmids bearing ARS 1501,
suggesting that REN1501 binds a protein important for replication
initiation. Only three elements found at origins are known to specifically
bind proteins. These include the ARS essential sequences and the Abf1p and
Rap1p DNA-binding sites. We show that the function of REN1501 at the origin
cannot be replaced by a Rap1p DNA-binding site or a site that binds the
transcriptional factor Gal4p and can only be partially substituted for by
an Abf1p recognition sequence. This implies that the role of the REN1501
element at the ARS 1501 origin is specific, and suggest that the frequency
of origin firing in eukaryotic cells may be regulated by origin-specific
enhancers.
ARTICLES
Functional analysis of a replication origin from Saccharomyces cerevisiae: identification of a new replication enhancer
Department of Microbiology, School of Medicine, The University of Connecticut Health Center, Farmington, CT 06030, USA.
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