Nucleic Acids Research, Vol 27, Issue 15 3079-3089, Copyright © 1999 by Oxford University Press
KP Reischmann, Z Zhang and GM Kapler
The Tetrahymena thermophila rDNA exists as a 21 kb palindromic
minichromosome with two initiation sites for replication in each half
palindrome. These sites localize to the imperfect, repeated 430 bp segments
that include the nucleosome-free domains 1 and 2 (D1 and D2). To determine
if the D1 and D2 segments act independently or in concert to control
initiation, stable DNA transformation assays were performed. Single domain
derivatives of the plasmid prD1 failed to support autonomous replication in
Tetrahymena. Instead, such constructs propagated exclusively by integration
into endogenous rDNA minichromosomes and displayed weak origin activity as
detected by 2D gel electrophoresis. D1/D1 and D2/D2 derivatives also
transformed Tetrahymena poorly, showing similar replication defects. Hence,
the D1 and D2 segments are functionally non-redundant and cooperate rather
than compete to control initiation. The observed replication defect was
greatly reduced in a plasmid derivative that undergoes palindrome formation
in Tetrahymena, suggesting that a compensatory mechanism overcomes this
replication block. Finally, using a transient replication assay, we present
evidence that phylogenetically-conserved type I elements directly regulate
DNA replication. Taken together, our data support a model in which
cooperative interactions between dispersed elements coordinately control
the initiation of DNA replication.
ARTICLES
Long range cooperative interactions regulate the initiation of replication in the Tetrahymena thermophila rDNA minichromosome
Department of Medical Biochemistry and Genetics, Texas A&M Health Science Center, College Station, TX 77843-1114, USA.
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