THE YEAST PROTEIN ENCODED BY PUB1 BINDS T-RICH SINGLE-STRANDED-DNA

We have characterized binding activities in yeast which recognise the T-rich strand of the yeast ARS consensus element and have purified two of these to homogeneity. One (ACBP-60) is detectable in both nuclear and whole cell extracts, while the other (ACBP-67) is apparent only af ter fractionation of extracts by heparin-sepharose chromatography. The major binding activity detected in nuclear extracts was purified on a sequence-specific DNA affinity column as a single polypeptide with ap parent mobility of 60kDa (ACBP-60). This protein cofractionates with n uclei, is present at several thousand copies per cell and has a K-d fo r the T-rich single strand of the ARS consensus between 10(-9) and 10( -10) M. Competition studies with simple nucleic acid polymers show tha t ACBP-60 has marginally higher affinity for poly dT(30) than for a 30 nt oligomer containing the T-rich strand of ARS 307, and approximatel y 10 fold higher affinity for poly rU. Internal sequence information o f purified p60 reveals identity with the open reading frames of genes PUB1 and RNP1 which encode polyuridylate binding protein(s). The secon d binding activity, ACBP-67, also binds specifically to the T-rich sin gle strand of the ARS consensus, but with considerably lower affinity than ACBP-60. Peptide sequence reveals that the 67kDa protein is ident ical to the major polyA binding protein in yeast, PAB1.