Allele-specific protein-DNA interactions between the single-stranded DNA-binding protein, ssA-TIBF, and DNA replication determinants in Tetrahymena

Type I elements are multifunctional, cia-acting determinants that regulate the initiation of DNA replication, replication fork movement and transcript ion of the Tetrahymena thermophila rDNA minichromosome. Previous studies id entified a protein, ssA-TIBF, that binds specifically to the A-rich strand of type I elements. Here, we examine interactions of ssA-TIBF with the wild -type C3 allele, and a natural variant, B rDNA, which manifests a defect in replication initiation and fork pausing. Purified ssA-TIBF is a homotetram er that binds one substrate molecule and contacts DNA via a single 24 kDa s ubunit. Both the A-rich and T-rich strands of type I elements are bound by ssA-TIBF, suggesting that this protein might stabilize replication origins in their unwound state. Nucleotides downstream of type I elements contribut e to DNA binding, with the extent of DNA-protein contact being greater for wild-type C3 rDNA compared to B rDNA. Allele-specific protein-DNA contacts also occur within the conserved type I element itself. Despite these differ ences, the binding affinities of ssA-TIBF for C3 and B rDNA substrates are indistinguishable. Consequently, the mode of DNA binding must account for a ny role ssA-TIBF might play in the regulation of rDNA replication. (C) 2000 Academic Press.