Architecture of the replication fork stalled at the 3 ' end of yeast ribosomal genes

Every unit of the rRNA gene cluster of Saccharomyces cerevisiae contains a unique site, termed the replication fork barrier (RFB), where progressing r eplication 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 clos ely spaced minor arrest sites were found. In the majority of molecules, the stalled lagging strand was completely processed and the discontinuously sy nthesized 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 endonu clease VII, an enzyme recognizing branched DNA molecules. This enzyme cleav ed predominantly in the newly synthesized homologous arms, thereby specific ally releasing the leading arm.