The mating-type-switch activating protein Sap1 is also required for replication fork blockage at the ribosomal DNA in Schizosaccharomyces pombe.
Eva Mejía-Ramírez, Alicia Sánchez-Gorostiaga, Dora B. Krimer, Jorge B. Schvarzman, Pablo Hernández
Biología Celular y del Desarr., CIB, CSIC, Ramiro de Maeztu, 9, Madrid, 28040, Spain
Schizosaccharomyces pombe ribosomal DNA (rDNA) contains three replication fork barriers (RFB1-3) located in the non-transcribed spacer, close to the 3'-end of the 35S gene. We have previously shown that RFB2 and RFB3 require binding of the transcription terminator factor Reb1p to two identical 17-bp recognition sequences that co-localized with these barriers. In the present work, we analyzed cis- and trans-acting factors involved in the remaining barrier, RFB1. A 78-bp sequence located 407-bp away from the 35S 3'-end was sufficient to induce fork blockage a in vivo plasmid replication assay. A protein of ~30 kDa that specifically binds to the 78-bp sequence was purified by affinity chromatography and identified as Sap1p by mass spectrometry. Sap1p is an essential, dimeric protein that binds the SAS1 site in the mat1 locus and is required for efficient mating-type switching. RFB1 and SAS1 show significant sequence homology. Mutations introduced in RFB1 that preclude formation of the Sap1p-RFB1 complex systematically abolish replication barrier function as analyze by 2D electrophoresis. These results strongly suggest that Sap1p is required for replication fork blockage at RFB1.