Nucleic Acids Research, Vol 24, Issue 5 816-823, Copyright © 1996 by Oxford University Press
RY Huang and D Kowalski
A yeast autonomously replicating sequence, ARS305, shares essential
components with a chromosome III replicator, ORI305. Known components
include an ARS consensus sequence (ACS) element, presumed to bind the
origin recognition complex (ORC), and a broad 3'-flanking sequence which
contains a DNA unwinding element. Here linker substitution mutagenesis of
ARS305 and analysis of plasmid mitotic stability identified three short
sequence elements within the broad 3'-flanking sequence. The major
functional element resides directly 3' of the ACS and the two remaining
elements reside further downstream, all within non-conserved ARS sequences.
To determine the contribution of the elements to replication origin
function in the chromosome, selected linker mutations were transplaced into
the ORI305 locus and two- dimensional gel electrophoresis was used to
analyze replication bubble formation and fork directions. Mutation of the
major functional element identified in the plasmid mitotic stability assay
inactivated replication origin function in the chromosome. Mutation of each
of the two remaining elements diminished both plasmid ARS and chromosomal
origin activities to similar levels. Thus multiple DNA elements identified
in the plasmid ARS are determinants of replication origin function in the
natural context of the chromosome. Comparison with two other genetically
defined chromosomal replicators reveals a conservation of functional
elements known to bind ORC, but no two replicators are identical in the
arrangement of elements downstream of ORC binding elements or in the extent
of functional sequences adjacent to the ACS.
ARTICLES
Multiple DNA elements in ARS305 determine replication origin activity in a yeast chromosome
Molecular and Cellular Biology Department, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
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