Three HRDC domains differentially modulate Deinococcus radiodurans RecQ DNA helicase biochemical activity
Three HRDC domains differentially modulate Deinococcus radiodurans RecQ DNA helicase biochemical activity
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DOI：
10.1074/jbc.m600097200
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发表时间：
2006-05-05
影响因子：
4.8
通讯作者：
Keck, JL
中科院分区：
文献类型：
--
作者：
Killoran, MP;Keck, JL
RecQ解旋酶是关键的基因组维持酶，在DNA复制、重组和修复中起作用。与几乎所有其他已确定的RecQ家族成员相比，来自耐辐射细菌耐辐射球菌的RecQ解旋酶在其C端编码三个“解旋酶和rnasedc末端”（HRDC）结构域。HRDC结构域与结构特异性核酸结合有关，其作用是将RecQ蛋白靶向特定的DNA结构；然而，迄今为止，只有具有单一HRDC结构域的RecQ蛋白被研究过。我们证明，HRDC结构域可以被蛋白水解地从D. radiodurans RecQ (DrRecQ) C端移除，每个结构域形成一个一致的结构域。利用这一观察结果作为指导，我们制作了一组缺乏HRDC结构域组合的重组DrRecQ变体，以研究它们的生化功能。在某些情况下，n端最末端的HRDC结构域对于高亲和力DNA结合和DNA的有效解绕至关重要。相反，更多的c端HRDC结构域会降低酶的DNA结合亲和力和DNA依赖性ATP水解率，并在结构特异性DNA解绕中发挥更复杂的作用。我们的研究结果表明，多个DrRecQ HRDC结构域已经进化为编码酶的DNA结合和调节功能。
RecQ helicases are key genome maintenance enzymes that function in DNA replication, recombination, and repair. In contrast to nearly every other identified RecQ family member, the RecQ helicase from the radioresistant bacterium Deinococcus radiodurans encodes three "Helicase and RNaseDC-terminal" (HRDC) domains at its C terminus. HRDC domains have been implicated in structure-specific nucleic acid binding with roles in targeting RecQ proteins to particular DNA structures; however, only RecQ proteins with single HRDC domains have been examined to date. We demonstrate that the HRDC domains can be proteolytically removed from the D. radiodurans RecQ (DrRecQ) C terminus, consistent with each forming a structural domain. Using this observation as a guide, we produced a panel of recombinant DrRecQ variants lacking combinations of its HRDC domains to investigate their biochemical functions. The N-terminal-most HRDC domain is shown to be critical for high affinity DNA binding and for efficient unwinding of DNA in some contexts. In contrast, the more C-terminal HRDC domains attenuate the DNA binding affinity and DNA-dependent ATP hydrolysis rate of the enzyme and play more complex roles in structure-specific DNA unwinding. Our results indicate that the multiple DrRecQ HRDC domains have evolved to encode DNA binding and regulatory functions in the enzyme.