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Molecular and Cellular Biology, August 2000, p. 5777-5787, Vol. 20, No. 15
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Architecture of the Replication Fork Stalled at the
3' End of Yeast Ribosomal Genes
Markus
Gruber,
Ralf Erik
Wellinger, and
José M.
Sogo*
Institute of Cell Biology, ETH
Hönggerberg, CH-8093 Zürich, Switzerland
Received 8 February 2000/Returned for modification 22 March
2000/Accepted 25 April 2000
Every unit of the rRNA gene cluster of Saccharomyces
cerevisiae contains a unique site, termed the replication fork
barrier (RFB), where progressing replication forks are stalled in a
polar manner. In this work, we determined the positions of the nascent strands at the RFB at nucleotide resolution. Within an
HpaI-HindIII fragment essential for the RFB, a
major and two closely spaced minor arrest sites were found. In the
majority of molecules, the stalled lagging strand was completely
processed and the discontinuously synthesized nascent lagging strand
was extended three bases farther than the continuously synthesized
leading strand. A model explaining these findings is presented. Our
analysis included for the first time the use of T4 endonuclease VII, an
enzyme recognizing branched DNA molecules. This enzyme cleaved
predominantly in the newly synthesized homologous arms, thereby
specifically releasing the leading arm.
*
Corresponding author. Mailing address: Institut
für Zellbiologie, ETH-Hönggerberg, CH-8093 Zürich,
Switzerland. Phone: 41 1 633 33 42. Fax: 41 1 633 10 69. E-mail:
sogo{at}cell.biol.ethz.ch.
Molecular and Cellular Biology, August 2000, p. 5777-5787, Vol. 20, No. 15
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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