YB-1 promotes strand separation in vitro of duplex DNA containing either mispaired bases or cisplatin modifications, exhibits endonucleolytic activities and binds several DNA repair proteins.

Abstract

YB-1 is a multifunctional protein involved in the regulation of transcription, translation, mRNA splicing and probably DNA repair. It contains a conserved cold shock domain and it binds strongly to inverted CCAAT box of different promoters. In this study, we have found that purified YB-1 oligomerizes readily in solutions to form trimers, hexamers and oligomers of 12 molecules. The presence of ATP changed the conformation of YB-1 in such a way that only dimers were detected by gel filtration analyses. Purified YB-1 can separate different DNA duplexes containing blunt ends, 5' or 3' recessed ends, or forked structures. This strand separation activity is increased on cisplatin-modified DNA or with duplex molecules containing mismatches. In addition to its exonuclease activity, YB-1 exhibits endonucleolytic activities in vitro. Finally, YB-1 affinity chromatography experiments have indicated that in addition to prespliceosome factors like nucleolin and ALY, YB-1 binds the DNA repair proteins MSH2, DNA polymerase delta, Ku80 and WRN proteins in vitro. Furthermore, immunofluorescence studies have shown that YB-1 re-localizes from the cytoplasm to nuclear areas containing either Ku80 or MSH2 proteins in human 293 embryonic kidney cells. These results suggest that YB-1 is involved in base excision and mismatch repair pathways.