Comparison of Dam tagging and chromatin immunoprecipitation as tools for the identification of the binding sites for S. pombe CENP-C.
We have established the identity of the Schizosaccharomyces pombe homologue of vertebrate CENP-C and Saccharomyces cerevisiae MIF2p and have used it to compare Dam tagging and chromatin immunoprecipitation (ChiP)as tools for the mapping of protein binding sites on DNA. ChiP shows that S. pombe CENP-C binds to the central core and inner repeats of the S. pombe centromere. It binds weakly, however, to the outer repeats. The binding pattern is thus similar to that of S. pombe CENP-A. Dam-tagged S. pombe CENP-C, however, methylates the entire centromere and 5 kb of flanking DNA. This comparison suggests that Dam tagging is less precise as a tool for mapping DNA binding sites than ChiP. We have also used the Dam tagging technique to address the question of whether there is any CENP-C binding to the ribosomal DNA in S. pombe and find none.
Holland
Sara
S
Institute of Genetics, School of Biology, Queen's Medical Centre, Nottingham, NG7 2UH, UK.
Ioannou
Dimitris
D
Haines
Steven
S
Brown
William R A
WR
D001324:Autoantigens; D001665:Binding Sites; D002503:Centromere / Q000502:physiology*; D047369:Chromatin Immunoprecipitation; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D004271:DNA, Fungal / Q000502:physiology; D004275:DNA, Ribosomal / Q000502:physiology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism*
D001324:Autoantigens; D002868:Chromosomal Proteins, Non-Histone; D004271:DNA, Fungal; D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; C083768:MIF2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C060905:centromere protein C; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)