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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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