Li K, Williams R S
Departments of Internal Medicine and Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-8573, USA.
J Biol Chem. 1997 Mar 28;272(13):8686-94. doi: 10.1074/jbc.272.13.8686.
We examined previously unexplored aspects of the tetramerization and single-stranded DNA (ssDNA) binding properties of native, precursor, and mutated forms of mitochondrial ssDNA-binding protein (mtSSB) from a mammalian organism (mouse). Tetramic forms of mtSSB reassemble spontaneously after thermal denaturation and undergo subunit exchange. Binding of mtSSB to ssDNA as a function of protein concentration is nonlinear, suggesting a concentration-dependent transition in intrinsic binding affinity and in the topology of the DNA-protein complex. The cleavable presequence at the amino terminus of the precursor form of mtSSB does not disrupt tetramer formation but has a specific inhibitory effect on DNA binding that is not seen in a fusion protein that substitutes a bulkier peptide moiety in this position. Mutated forms of mtSSB bearing amino acid substitutions at highly conserved amino acid positions exhibit subtle or severe defects in ssDNA binding activity and/or tetramerization, even when assembled into heterotetramers in combination with wild-type mtSSB monomers. These experiments provide new insights into structural and functional properties of mammalian mtSSB and have implications for the pathogenesis of human diseases resulting from defects in mtDNA replication.
我们研究了来自哺乳动物（小鼠）的线粒体单链DNA结合蛋白（mtSSB）的天然、前体和突变形式在四聚化及单链DNA（ssDNA）结合特性方面此前未被探索的内容。mtSSB的四聚体形式在热变性后会自发重新组装并发生亚基交换。mtSSB与ssDNA的结合随蛋白质浓度的变化呈非线性，这表明内在结合亲和力以及DNA-蛋白质复合物的拓扑结构存在浓度依赖性转变。mtSSB前体形式氨基末端的可切割前导序列不会破坏四聚体的形成，但对DNA结合具有特定的抑制作用，而在该位置替换了更大肽部分的融合蛋白中未观察到这种作用。在高度保守氨基酸位置带有氨基酸替代的mtSSB突变形式，即使与野生型mtSSB单体组装成异源四聚体，在ssDNA结合活性和/或四聚化方面也表现出细微或严重的缺陷。这些实验为哺乳动物mtSSB的结构和功能特性提供了新的见解，并对由线粒体DNA复制缺陷导致的人类疾病的发病机制具有启示意义。
