The Replication Fork Barrier Site Forms a Unique Structure with Fob1p and Inhibits the Replication Fork

Abstract

The replication fork barrier site (RFB) is an ∼100-bp DNA sequence located near the 3′ end of the rRNA genes in the yeast Saccharomyces cerevisiae. The gene FOB1 is required for this RFB activity. FOB1 is also necessary for recombination in the ribosomal DNA (rDNA), including increase and decrease of rDNA repeat copy number, production of extrachromosomal rDNA circles, and possibly homogenization of the repeats. Despite the central role that Foblp plays in both replication fork blocking and rDNA recombination, the molecular mechanism by which Fob1p mediates these activities has not been determined. Here, I show by using chromatin immunoprecipitation, gel shift, footprinting, and atomic force microscopy assays that Fob1p directly binds to the RFB. Fob1p binds to two separated sequences in the RFB. A predicted zinc finger motif in Fob1p was shown to be essential for the RFB binding, replication fork blocking, and rDNA recombination activities. The RFB seems to wrap around Fob1p, and this wrapping structure may be important for function in the rDNA repeats.

ACKNOWLEDGMENTS

I thank M. Nomura (UCI, Irvine, Calif.) and T. Horiuchi (NIBB, Okazaki, Japan) for critical reading of the manuscript. I thank Y. Kamimura and H. Araki (National Institute of Genetics, Mishima, Japan) for technical advice on the ChIP assay, T. Yasuda and F. Hanaoka (RIKEN, Wako, Japan) for technical advice on the gel shift assay, and A. Ganley of our laboratory for preparation of the manuscript and discussion. Strain YK9 and pEG(KT) were kindly provided by Y. Kawasaki (Osaka University, Osaka, Japan).

This work was supported in part by grants-in-aid for scientific research 13141205, 13480234, and 14380332 from the Ministry of Education, Science and Culture, Japan; by a grant from the Ministry of Health and Welfare, Japan; and by a grant from the Human Frontier Science Organization, France.