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 l imited set of proteins able to bind to RNA,DNA hybrids in both Xenopus oocy te extracts and human macrophage extracts. Resulting patterns of crosslinke d proteins were highly dependent on the strand (DNA or RNA) that was substi tuted. 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 -strand ed 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 met hodology, we have detected proteins that bind to short oligodeoxyribonucleo tides. Although we have essentially detected in Xenopus oocytes one promine nt protein of approximate to 75 kDa, corresponding to replication protein A (RPA) whatever the oligonucleotide used, the patterns obtained with extrac ts of human macrophages were more complex and dependent on the oligonucleot ide used. If a protein corresponding to RPA was observed most of the time, other crosslinks of similar or sometimes higher intensity were also detecte d. Interestingly, among these, one protein of 35 kDa appears paradoxically to bind and crosslink to a dodecamer but not to an octadecamer containing t he same sequence placed either at its 3'-end or 5'-end.