Nucleic Acids Research, Vol 27, Issue 19 3921-3930, Copyright © 1999 by Oxford University Press
CA Miller, RM Umek and D Kowalski
Ribosomal DNA (rDNA) replication origins of Saccharomyces cerevisiae are
known to function inefficiently, both in the context of the tandem rDNA
repeats in the chromosome and as single copy autonomously replicating
sequences (ARSs) in plasmids. Here we examined components of the rDNA ARS
that might contribute to inefficient extrachromosomal replication. Like the
efficient H4 ARS, the rDNA ARS requires a match to the 11 bp ARS consensus
sequence (ACS) and a broad non-conserved region that may contain multiple
elements, including a DNA unwinding element (DUE). Using a
single-strand-specific nuclease hypersensitivity assay and by determining
the superhelical density required for stable DNA unwinding, we found that
the DNA of the rDNA ARS is not as easily unwound as the H4 ARS. Unwinding
of the rDNA ARS required additional energy, similar to the unwinding of
mutations in the H4 ARS that stabilize the double helix in the DUE region
and impair replication. In vivo extrachromosomal replication of the rDNA
ARS was cold sensitive, like H4 ARS mutants that require additional energy
to unwind the DUE region but unlike the easily unwound, wild-type H4 ARS.
Impairment of replication function at reduced temperature suggests that the
elevated energy requirement for DNA unwinding inherent in the wild-type
rDNA ARS contributes to inefficient replication function. We also examined
the essential ACS match in the rDNA ARS, which is known to be imperfect at
one position. A point mutation in the essential ACS that corrects the
imperfect match increased the efficiency of extrachromosomal replication.
Our results reveal that the essential ACS element and DNA unwinding in the
rDNA ARS are naturally impaired, suggesting that inefficient function of
the rDNA replication origin has a biological purpose.
ARTICLES
The inefficient replication origin from yeast ribosomal DNA is naturally impaired in the ARS consensus sequence and in DNA unwinding
Cancer Genetics Department, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
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