Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA.
Nucleic Acids Res. 2012 Oct;40(18):9233-43. doi: 10.1093/nar/gks688. Epub 2012 Jul 24.
Bacterial and archaeal topoisomerase I display selectivity for a cytosine base 4 nt upstream from the DNA cleavage site. Recently, the solved crystal structure of Escherichia coli topoisomerase I covalently linked to a single-stranded oligonucleotide revealed that R169 and R173 interact with the cytosine base at the -4 position via hydrogen bonds while the phenol ring of Y177 wedges between the bases at the -4 and the -5 position. Substituting R169 to alanine changed the selectivity of the enzyme for the base at the -4 position from a cytosine to an adenine. The R173A mutant displayed similar sequence selectivity as the wild-type enzyme, but weaker cleavage and relaxation activity. Mutation of Y177 to serine or alanine rendered the enzyme inactive. Although mutation of each of these residues led to different outcomes, R169, R173 and Y177 work together to interact with a cytosine base at the -4 position to facilitate DNA cleavage. These strictly conserved residues might act after initial substrate binding as a Molecular Ruler to form a protein-DNA complex with the scissile phosphate positioned at the active site for optimal DNA cleavage by the tyrosine hydroxyl nucleophile to facilitate DNA cleavage in the reaction pathway.
细菌和古菌拓扑异构酶 I 对 DNA 切割位点上游 4 个核苷酸的胞嘧啶碱基具有选择性。最近，解出的与单链寡核苷酸共价连接的大肠杆菌拓扑异构酶 I 的晶体结构表明，R169 和 R173 通过氢键与 -4 位的胞嘧啶碱基相互作用，而 Y177 的酚环则楔入 -4 位和 -5 位碱基之间。将 R169 突变为丙氨酸会改变酶对 -4 位碱基的选择性，使酶对胞嘧啶的选择性变为腺嘌呤。R173A 突变体显示出与野生型酶相似的序列选择性，但切割和松弛活性较弱。将 Y177 突变为丝氨酸或丙氨酸会使酶失活。尽管这些残基的突变导致了不同的结果，但 R169、R173 和 Y177 共同作用于 -4 位的胞嘧啶碱基，以促进 DNA 切割。这些严格保守的残基可能在初始底物结合后作为分子标尺发挥作用，形成带有可切割磷酸的蛋白质-DNA 复合物，使酪氨酸羟基亲核试剂在活性位点定位，以促进反应途径中的 DNA 切割。
