The DNA binding properties of the Escherichia coli RecQ helicase
The DNA binding properties of the Escherichia coli RecQ helicase
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DOI：
10.1074/jbc.m311272200
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发表时间：
2004-02-20
影响因子：
4.8
通讯作者：
Xi, XG
中科院分区：
文献类型：
--
作者：
Dou, SX;Wang, PY;Xi, XG
RecQ解旋酶家族从细菌到人高度保守，并且在基因组完整性的保存中起保守作用。它在人类细胞中的缺乏导致与过早衰老和癌症相关的明显的基因组不稳定性。为了确定大肠杆菌RecQ解旋酶与DNA相互作用的热力学参数，已使用热力学严格的荧光滴定技术进行了平衡结合研究。荧光素标记的寡核苷酸的稳态荧光各向异性测量显示，RecQ解旋酶与DNA结合的表观结合化学计量比为1蛋白单体/10个核苷酸。在AMPPNP（腺苷5 '-（β，γ-亚氨基）三磷酸）或ADP存在下，该化学计量没有改变。RecQ解旋酶与不同长度的寡核苷酸在广泛的pH、NaCl和核酸浓度范围内相互作用的分析表明，RecQ解旋酶具有单一强DNA结合位点，在25 ℃下的结合常数为K = 6.7 +/- 0.95 x 10（6）M-1，协同性参数为ω = 25.5 +/- 1.2。单链DNA和双链DNA都竞争性地结合到同一位点。内在亲和力是盐依赖性的，DNA-解旋酶复合物的形成伴随着3-4个离子的净释放。仅在1.5 mm AMPPNP存在下观察到核苷酸辅因子对RecQ与DNA结合的变构效应，而AMPPNP和ADP在大范围的核苷酸辅因子浓度下对双链DNA结合没有可检测的影响。
The RecQ helicase family is highly conserved from bacteria to men and plays a conserved role in the preservation of genome integrity. Its deficiency in human cells leads to a marked genomic instability that is associated with premature aging and cancer. To determine the thermodynamic parameters for the interaction of Escherichia coli RecQ helicase with DNA, equilibrium binding studies have been performed using the thermodynamic rigorous fluorescence titration technique. Steady-state fluorescence anisotropy measurements of fluorescein-labeled oligonucleotides revealed that RecQ helicase bound to DNA with an apparent binding stoichiometry of 1 protein monomer/10 nucleotides. This stoichiometry was not altered in the presence of AMPPNP (adenosine 5'-(beta,gamma-imido) triphosphate) or ADP. Analyses of RecQ helicase interactions with oligonucleotides of different lengths over a wide range of pH, NaCl, and nucleic acid concentrations indicate that the RecQ helicase has a single strong DNA binding site with an association constant at 25 degreesC of K = 6.7 +/- 0.95 x 10(6) M-1 and a cooperativity parameter of omega = 25.5 +/- 1.2. Both single-stranded DNA and double-stranded DNA bind competitively to the same site. The intrinsic affinities are salt-dependent, and the formation of DNA-helicase complex is accompanied by a net release of 3-4 ions. Allosteric effects of nucleotide cofactors on RecQ binding to DNA were observed only for single-stranded DNA in the presence of 1.5 mm AMPPNP, whereas both AMPPNP and ADP had no detectable effect on double-stranded DNA binding over a large range of nucleotide cofactor concentrations.