Detection of proteins binding to short RNA.DNA hybrids or short antisense oligonucleotides in Xenopus laevis oocytes and human macrophage cell extracts by photoaffinity radiolabeling.
Using a 12 base pair RNA.DNA hybrid, substituted with bromouracil on either the RNA or DNA strand, we have detected by photoaffinity radiolabeling a limited set of proteins able to bind to RNA.DNA hybrids in both Xenopus oocyte extracts and human macrophage extracts. Resulting patterns of crosslinked proteins were highly dependent on the strand (DNA or RNA) that was substituted. With one exception, none of the proteins investigated in competition experiments was found to be absolutely specific for RNA.DNA hybrids, as at least one other nucleic acid, either single-stranded DNA or single-stranded RNA, was found to compete efficiently. None of the proteins detected in this assay correspond to the size expected for RNases H. Using the same methodology, we have detected proteins that bind to short oligodeoxyribonucleotides. Although we have essentially detected in Xenopus oocytes one prominent protein of approximately 75 kDa, corresponding to replication protein A (RPA) whatever the oligonucleotide used, the patterns obtained with extracts of human macrophages were more complex and dependent on the oligonucleotide used. If a protein corresponding to RPA was observed most of the time, other crosslinks of similar or sometimes higher intensity were also detected. Interestingly, among these, one protein of 35 kDa appears paradoxically to bind and crosslink to a dodecamer but not to an octadecamer containing the same sequence placed either at its 3'-end or 5'-end.
Revers
F
F
Laboratoire de Biophysique Moléculaire, INSERM U386, Université Victor Segalen Bordeaux II, France.
Cario
M
M
Cao
T L
TL
Cazenave
C
C
D000818:Animals; D002457:Cell Extracts; D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008264:Macrophages; D009693:Nucleic Acid Hybridization; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000378:metabolism*; D009865:Oocytes; D019920:Photoaffinity Labels; D011485:Protein Binding; D012313:RNA / Q000378:metabolism*; D051716:Replication Protein A; D014982:Xenopus laevis
D002457:Cell Extracts; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D016376:Oligonucleotides, Antisense; D019920:Photoaffinity Labels; C497167:RPA1 protein, human; D051716:Replication Protein A; D012313:RNA; D004247:DNA