Saha S, Kapler G M
College Station, Texas A&M Health Science Center, TX, 77843-1114, USA.
J Mol Biol. 2000 Jan 21;295(3):423-39. doi: 10.1006/jmbi.1999.3365.
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.
I型元件是多功能的顺式作用决定子，可调节嗜热四膜虫rDNA微型染色体的DNA复制起始、复制叉移动和转录。先前的研究鉴定出一种蛋白质，即ssA-TIBF，它能特异性结合I型元件富含A的链。在这里，我们研究了ssA-TIBF与野生型C3等位基因以及一种天然变体B rDNA之间的相互作用，B rDNA在复制起始和叉停顿方面表现出缺陷。纯化的ssA-TIBF是一种同四聚体，它结合一个底物分子，并通过单个24 kDa亚基与DNA接触。I型元件富含A和富含T的链均被ssA-TIBF结合，这表明该蛋白质可能在其解旋状态下稳定复制起点。I型元件下游的核苷酸有助于DNA结合，与B rDNA相比，野生型C3 rDNA的DNA-蛋白质接触程度更大。等位基因特异性的蛋白质-DNA接触也发生在保守的I型元件本身内部。尽管存在这些差异，但ssA-TIBF对C3和B rDNA底物的结合亲和力并无区别。因此，DNA结合模式必须解释ssA-TIBF在rDNA复制调控中可能发挥的任何作用。
