Biochemical studies of yeast replication or gin binding protein sand initiate complexes

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South-western screening of a glutathione-S-transferase fusion protein library constructed from the yeast Saccharomyces cerevisiae genomic DNA lead to isolation of core T-rich strand binding protein (CTBP) clones that bound to single-stranded DNA containing the T-rich strand of the 11 base-pair core sequence of autonomously replicating sequences. One of these clones, CTBP1, contains a portion of previously described RBP1 which is an RNA- and single-stranded DNA binding protein of Saccharomyces cerevisiae. GST-CTBP1 fusion protein binds exclusively to the T-rich strand of the core sequence with apparent dissociation constant of 5*10^<-9> M but not to the A-rich strand or double-strand of the same sequence. Mutations within the core which reduce the number of T or C residues decrease the affinity to this protein. In keeping with this, binding to the core sequence is efficiently competed by poly [dT] or ply [dT-dC] as well as by poly [dC] albeit to a lesser extent, but not by poly [dA] or … More poly [dG] to any significant extent. Among polyribonucleic acids, it binds to poly [U] and poly [T] with affinity similar to but not greater than that of poly [dT] and poly [dC], respectively. Our results indicate that CTBP1 is a polydeoxypyrimidine binding protein of Saccharomyces cerevisiae. CTBP1 contains two sets of an RNA recognition motif (RRM) and a glutamine-stretch. The binding affinity of the N-terminal or C-terminal set containing one RRM and one glutamine stretch is nearly two orders of magnitude lower than that of the "wild-type" CTBP1 containing both sets. The isolated N-terminal or C-terminal RRM alone (RRM1 and RRM2, respectively) is sufficient for binding nucleic acids with the binding specificity similar to that of the "wild-type", although binding affinity of the isolated RRM2 is nearly two orders of magnitude lower than that of RRM1. Our results indicate that two RRMs present in CTBP1/RBP1 have differential binding affinity and that its high affinity to polydeoxypyrimidine results from synergy between two lower affinity RRMs. Less