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, cis-acting determinants that regulate the initiation of DNA replication, replication fork movement and transcription of the Tetrahymena thermophila rDNA minichromosome. Previous studies identified 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 homotetramer that binds one substrate molecule and contacts DNA via a single 24 kDa subunit. 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 contribute 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 differences, the binding affinities of ssA-TIBF for C3 and B rDNA substrates are indistinguishable. Consequently, the mode of DNA binding must account for any role ssA-TIBF might play in the regulation of rDNA replication.
Saha
S
S
College Station, Texas A&M Health Science Center, TX, 77843-1114, USA.
Kapler
G M
GM
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D001704:Biopolymers; D004261:DNA Replication / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D015800:Protozoan Proteins; D016808:Tetrahymena thermophila / Q000235:genetics*
D001704:Biopolymers; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D015800:Protozoan Proteins; C404082:ssA-TIBF protein, Tetrahymena thermophila