Energetics of DNA end binding by E.coli RecBC and RecBCD helicases indicate loop formation in the 3'-single-stranded DNA tail.
Energetics of DNA end binding by E.coli RecBC and RecBCD helicases indicate loop formation in the 3'-single-stranded DNA tail.
复制标题
大肠杆菌 RecBC 和 RecBCD 解旋酶与 DNA 末端结合的能量表明 3-单链 DNA 尾部形成环。
DOI：
10.1016/j.jmb.2005.07.056
复制
发表时间：
2005
影响因子：
5.6
通讯作者：
Lohman,TimothyM
中科院分区：
文献类型：
--
作者：
Wong,CJason;Lucius,AaronL;Lohman,TimothyM
我们研究了大肠杆菌RecBC和RecBCD解旋酶与双链体DNA末端的平衡结合，双链体DNA末端具有预先存在的不同长度（n = 0至20个核苷酸）的单链（ss）DNA（（dT）n）尾，以确定3 ′和5 ′单链对复合物形成的能量学的贡献。通过在其末端用Cy3荧光团标记的参考DNA的荧光增强来监测蛋白质结合。通过与Cy3标记的参考DNA的竞争滴定来检查与未标记的DNA的结合。RecBC和RecBCD的亲和力随着3 ′-（dT）ntail长度从0增加到6个核苷酸而增加，但随后随着3 ′-（dT）ntail长度从6增加到20个核苷酸而急剧降低。用RecBC进行的等温滴定量热实验表明，结合焓为负，并且随着3 ′-（dT）n尾长度的增加而增加，直到n = 6个核苷酸，但当n ≥ 6时保持恒定。因此，当n ≥ 6时，3 ′-（dT）n尾长度的结合亲和力降低是由于不利的熵贡献。RecBC与3 '和5'端均带有（dT）6尾的双链DNA结合最佳，而RecBCD则更喜欢带有3 '-（dT）6和5'-（dT）10尾的双链DNA。这些数据表明，RecBC和RecBCD解旋酶在ATP不存在的情况下结合到钝的DNA双链体末端时可以使六个碱基对不稳定或"熔化"。这些结果也首次证明了当RecBC或RecBCD与含有n ≥ 6个核苷酸的预先形成的3 ′-ssDNA尾的DNA双链体结合时，3 ′-ssDNA尾中可以形成环。这些环可能代表了那些假设在DNA解旋过程中包含在解旋的3 ′ ss-DNA尾中的Chi位点与RecC亚基相互作用后形成的环。
We examined the equilibrium binding of Escherichia coli RecBC and RecBCD helicases to duplex DNA ends possessing pre-existing single-stranded (ss) DNA ((dT)n) tails varying in length (n=0 to 20 nucleotides) in order to determine the contributions of both the 3′ and 5′ single strands to the energetics of complex formation. Protein binding was monitored by the fluorescence enhancement of a reference DNA labeled at its end with a Cy3 fluorophore. Binding to unlabeled DNA was examined by competition titrations with the Cy3-labeled reference DNA. The affinities of both RecBC and RecBCD increase as the 3′-(dT)ntail length increases from zero to six nucleotides, but then decrease dramatically as the 3′-(dT)ntail length increases from six to 20 nucleotides. Isothermal titration calorimetry experiments with RecBC show that the binding enthalpy is negative and increases in magnitude with increasing 3′-(dT)ntail length up to n=6 nucleotides, but remains constant for n ≥6. Hence, the decrease in binding affinity for 3′-(dT)ntail lengths with n ≥6 is due to an unfavorable entropic contribution. RecBC binds optimally to duplex DNA with (dT)6tails on both the 3′ and 5′-ends while RecBCD prefers duplex DNA with 3′-(dT)6and 5′-(dT)10tails. These data suggest that both RecBC and RecBCD helicases can destabilize or “melt out” six base-pairs upon binding to a blunt DNA duplex end in the absence of ATP. These results also provide the first evidence that a loop in the 3′-ssDNA tail can form upon binding of RecBC or RecBCD with DNA duplexes containing a pre-formed 3′-ssDNA tail with n ≥6 nucleotides. Such loops may be representative of those hypothesized to form upon interaction of a Chi site contained within the unwound 3′ ss-DNA tail with the RecC subunit during DNA unwinding.