Two steps forward, one step back: determining XPD helicase mechanism by single-molecule fluorescence and high-resolution optical tweezers.
Two steps forward, one step back: determining XPD helicase mechanism by single-molecule fluorescence and high-resolution optical tweezers.
复制标题
DOI：
10.1016/j.dnarep.2014.01.013
复制
发表时间：
2014-08
期刊：
影响因子：
3.8
通讯作者：
Spies, Maria
中科院分区：
文献类型：
--
作者：
Spies, Maria
关键词：
XPD样解旋酶构成了一个重要的DNA解旋酶家族，对于基因组维持的许多方面至关重要。这些酶具有独特的结构特征，由铁硫（FeS）簇稳定的辅助结构域，以及DNA易位和双链体解旋的5′-3′极性。生化分析以及两种单分子方法，全内反射荧光显微镜和高分辨率光镊，已经显示了XPD解旋酶的独特结构特征及其特定的底物相互作用模式如何调整解旋酶的特定细胞功能并塑造其分子机制。FeS结构域形成双链体分离楔，并有助于扩展DNA结合位点。在该位点内的相互作用将解旋酶定位在使双链体解旋的方向上，控制解旋酶速率，并验证易位链的完整性。与其细胞作用一致，XPD的持续合成能力是有限的，并且由特异质步进动力学定义。DNA双链体分离发生在单碱基对步骤中，被频繁的反向步骤和蛋白质-DNA复合物的构象重排打断。因此，分离的解旋酶主要稳定自发的碱基对打开，并表现出有限的解旋稳定DNA双链体的能力。同源ssDNA结合蛋白的存在通过使上游dsDNA不稳定以及通过捕获解缠绕的链将XPD转化为有力的解旋酶。值得注意的是，这两种蛋白质可以共存于同一条DNA链上，而不会竞争结合。XPD解旋机制的当前模型将被讨论沿着与解旋酶相互作用伙伴和独特的生物医学重要的XPD样解旋酶如FANCJ（BACH 1）、RTEL 1和CHLR 1（DDX 11）的独特特征对该机制的可能修改。
XPD-like helicases constitute a prominent DNA helicase family critical for many aspects of genome maintenance. These enzymes share a unique structural feature, an auxiliary domain stabilized by an iron-sulphur (FeS) cluster, and a 5′-3′ polarity of DNA translocation and duplex unwinding. Biochemical analyses alongside two single-molecule approaches, total internal reflection fluorescence microscopy and high-resolution optical tweezers, have shown how the unique structural features of XPD helicase and its specific patterns of substrate interactions tune the helicase for its specific cellular function and shape its molecular mechanism. The FeS domain forms a duplex separation wedge and contributes to an extended DNA binding site. Interaction within this site position the helicase in an orientation to unwind the duplex, control the helicase rate, and verify the integrity of the translocating strand. Consistent with its cellular role, processivity of XPD is limited and is defined by an idiosyncratic stepping kinetics. DNA duplex separation occurs in single base pair steps punctuated by frequent backward steps and conformational rearrangements of the protein-DNA complex. As such, the helicase in isolation mainly stabilizes spontaneous base pair opening and exhibits a limited ability to unwind stable DNA duplexes. The presence of a cognate ssDNA binding protein converts XPD into a vigorous helicase by destabilizing the upstream dsDNA as well as by trapping the unwound strands. Remarkably, the two proteins can co-exist on the same DNA strand without competing for binding. The current model of the XPD unwinding mechanism will be discussed along with possible modifications to this mechanism by the helicase interacting partners and unique features of such bio-medically important XPD-like helicases as FANCJ (BACH1), RTEL1 and CHLR1 (DDX11).
登录
查看更多内容
影响因子：
11.4
作者：
Bochkarev, A;Bochkareva, E;Edwards, AM
通讯作者：
Edwards, AM
影响因子：
56.9
作者：
Ali, JA;Lohman, TM
通讯作者：
Lohman, TM
影响因子：
4.8
作者：
Buechner, Claudia N.;Heil, Korbinian;Tessmer, Ingrid
通讯作者：
Tessmer, Ingrid
影响因子：
64.8
作者：
Chen, J;Larochelle, S;Suter, B
通讯作者：
Suter, B
影响因子：
30.8
作者：
通讯作者：
--