Molecular architecture of a eukaryotic DNA replication terminus-terminator protein complex.
DNA replication forks pause at programmed fork barriers within nontranscribed regions of the ribosomal DNA (rDNA) genes of many eukaryotes to coordinate and regulate replication, transcription, and recombination. The mechanism of eukaryotic fork arrest remains unknown. In Schizosaccharomyces pombe, the promiscuous DNA binding protein Sap1 not only causes polar fork arrest at the rDNA fork barrier Ter1 but also regulates mat1 imprinting at SAS1 without fork pausing. Towards an understanding of eukaryotic fork arrest, we probed the interactions of Sap1 with Ter1 as contrasted with SAS1. The Sap1 dimer bound Ter1 with high affinity at one face of the DNA, contacting successive major grooves. The complex displayed translational symmetry. In contrast, Sap1 subunits approached SAS1 from opposite helical faces, forming a low-affinity complex with mirror image rotational symmetry. The alternate symmetries were reflected in distinct Sap1-induced helical distortions. Importantly, modulating protein-DNA interactions of the fork-proximal Sap1 subunit with the nonnatural binding site DR2 affected blocking efficiency without changes in binding affinity or binding mode but with alterations in Sap1-induced DNA distortion. The results reveal that Sap1-DNA affinity alone is insufficient to account for fork arrest and suggest that Sap1 binding-induced structural changes may result in formation of a competent fork-blocking complex.
Krings
Gregor
G
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA.
Bastia
Deepak
D
D001483:Base Sequence; D004261:DNA Replication; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012313:RNA; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D019098:Telomerase
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D021122:Protein Subunits; C516545:SAS1 protein, S pombe; C085981:Sap1 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; C516544:Ter1 telomerase subunit, S pombe; D012313:RNA; D019098:Telomerase