The mating type switch-activating protein Sap1 Is required for replication fork arrest at the rRNA genes of fission yeast.
Schizosaccharomyces pombe rRNA genes contain three replication fork barriers (RFB1-3) located in the nontranscribed spacer. RFB2 and RFB3 require binding of the transcription terminator factor Reb1p to two identical recognition sequences that colocalize with these barriers. RFB1, which is the strongest of the three barriers, functions in a Reb1p-independent manner, and cognate DNA-binding proteins for this barrier have not been identified yet. Here we functionally define RFB1 within a 78-bp sequence located near the 3' end of the rRNA coding region. A protein that specifically binds to this sequence was purified by affinity chromatography and identified as Sap1p by mass spectrometry. Specific binding to RFB1 was confirmed by using Sap1p expressed in Escherichia coli. Sap1p is essential for viability and is required for efficient mating-type switching. Mutations in RFB1 that precluded formation of the Sap1p-RFB1 complex systematically abolished replication barrier function, indicating that Sap1p is required for replication fork blockage at RFB1.
Mejía-Ramírez
Eva
E
Departamento de Biología Celular y del Desarrollo, Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain.
Sánchez-Gorostiaga
Alicia
A
Krimer
Dora B
DB
Schvartzman
Jorge B
JB
Hernández
Pablo
P
D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D004247:DNA / Q000737:chemistry; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000378:metabolism; D005800:Genes, Fungal; D013058:Mass Spectrometry; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012335:RNA, Ribosomal / Q000737:chemistry* / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics* / Q000502:physiology*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D012335:RNA, Ribosomal; C085981:Sap1 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; C512907:reb1 protein, S pombe; D004247:DNA