Analysis of the RAP1 protein binding to homogeneous telomeric repeats in Saccharomyces castellii.
The repressor activator protein 1 (RAP1) plays a role in telomere structure and function inS. cerevisiae. Here, the RAP1 homologue was identified and cloned from the budding yeast Saccharomyces castellii (scasRAP1). The scasRAP1 gene encodes a protein of 826 amino acids and shares an overall high degree of similarity with the S. cerevisiae RAP1 (scerRAP1). We demonstrate that the scasRAP1 is able to complement scerRAP1 in temperature-sensitive S. cerevisiae strains and is able to function as a regulator to maintain the original telomere lengths. Binding analyses of the E. coli-expressed scasRAP1 protein demonstrate that it needs two consecutive telomeric repeats in order to bind the S. castellii telomeric DNA sequences, and that it binds adjacent sites having a 16 bp centre-to-centre spacing. The binding affinity to telomeric DNA of several other yeasts is similar to that of scerRap1p. However, in contrast to scerRap1p, scasRap1p was found to bind the human telomeric sequence. Moreover, the scasRap1p was found to incorporate a variant repeat in its binding to the otherwise homogeneous telomeric DNA of S. castellii. This ability to bind various sites differing in DNA sequence indicates a high degree of adjustability in the binding of scasRap1p to DNA.
Wahlin
Johan
J
Department of Molecular Genetics, Lund University, Sölvegatan 29, S-223 62 Lund, Sweden.
Cohn
Marita
M
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012440:Saccharomyces / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D016615:Telomere / Q000378:metabolism*; D034501:Telomere-Binding Proteins
D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D034501:Telomere-Binding Proteins; C074461:RIF1 protein, S cerevisiae; D003850:Deoxyribonuclease I