Abstract
Eukaryotic DNA replication forks stall at natural replication fork barriers or Ter sites located within the ribosomal DNA (rDNA) intergenic spacer regions during unperturbed DNA replication. The rDNA intergenic spacer of the fission yeast Schizosaccharomyces pombe contains four polar or orientation-specific fork barriers, Ter1-3 and RFP4. Whereas the transcription terminator Reb1p binds Ter2 and Ter3 to arrest replication, the factor(s) responsible for fork arrest at Ter1 and RFP4 remain unknown. Using linker scanning mutagenesis, we have narrowed down minimal Ter1 to 21 bp. Sequence analysis revealed the presence of a consensus binding motif for the essential switch-activating and genome-stabilizing protein Sap1p within this region. Recombinant Sap1p bound Ter1 with high specificity, and endogenous Ter1 binding activity contained Sap1p and comigrated with the Sap1p-Ter1 complex. Circular permutation analysis suggested that Sap1p bends Ter1 and SAS1 upon binding. Targeted mutational analysis revealed that Ter1 mutations, which prevent Sap1p binding in vitro, are defective for replication fork arrest in vivo, whereas mutations that do not affect Sap1p binding remain competent to arrest replication. The results confirm the hypothesis that the chromatin organizer Sap1p binds site-specifically to genomic regions other than SAS1 and support the notion that Sap1p binds the rDNA fork barrier Ter1 to cause polar replication fork arrest at this site but not at SAS1.
Highlights
Conserved from yeast to humans [11,12,13,14,15,16,17]
Minimal Ter1 Activity Is Functionally Contained within a 21-bp Region of the S. pombe ribosomal DNA (rDNA)—We have previously identified four replication fork barriers, Ter1–3 and RFP4 within the S. pombe intergenic rDNA region (Fig. 1A)
Ter1–3 were all found to require the intra-S phase checkpoint proteins Swi1p and Swi3p for activity [13], the initiating cause for polar fork pausing was only identified for Ter2 and Ter3, which correspond to binding sites for the RNA polymerase I transcription terminator Reb1p [13, 25, 36]
Summary
Strains—S. pombe SP976 (h90, ade6-M210, ura4-D18, leu1-32) was used for all yeast experiments. Plasmids—Various overlapping fragments were amplified by PCR from Ter1-containing pIS8 [13] and cloned into the unique BamHI site of pIRT2. 3 fmol of labeled double-stranded oligonucleotide probes were incubated with the indicated amounts of His6-Sap or S. pombe crude whole cell extracts in 20 mM Tris, pH 8, 50 mM NaCl, 5% glycerol, and either 100 ng or 1 g of sheared salmon sperm DNA, respectively, for 5 min at room temperature. Bound and free probes were electrophoresed through 6% native polyacrylamide gels containing 2.5% glycerol using 0.5ϫ Tris borate/EDTA running buffer. Supershift assays were performed in a similar manner as gel shift assays, except that bound complexes were further incubated with indicated dilutions of anti-Sap1p antiserum directed at the N terminus of Sap1p (generously provided by Dr Benoit Arcangioli, Institute Pasteur, Paris Cedex, France) for 10 min at room temperature prior to electrophoresis. Relative mobilities were calculated for the free and bound probes by measuring the distance each migrated from the wells
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