Holliday junction resolution is modulated by archaeal chromatin components in vitro
Holliday junction resolution is modulated by archaeal chromatin components in vitro
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DOI：
10.1074/jbc.m109496200
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发表时间：
2002-01-25
影响因子：
4.8
通讯作者：
White, MF
中科院分区：
文献类型：
--
作者：
Kvaratskhelia, M;Wardleworth, BN;White, MF
Holliday连接解析酶Me在古细菌中是保守的，并且可能在同源重组途径中起类似于大肠杆菌RuvC的作用。Me特异性识别四向DNA连接，在没有序列偏好的情况下切割它们以产生重组DNA双链体产物。Hjc在结合的DNA连接上施加X形全局构象，并扭曲切割点周围的碱基堆积，连接中心3'的三个核苷酸。我们表明，我是自抑制单营业额测定条件下，这可以通过添加竞争对手的双链体DNA或建筑双链DNA结合蛋白Sso 7 d（即通过近似体内条件更密切）来缓解。使用等温滴定量热法和荧光共振能量转移的组合，我们证明了多个Hjc二聚体可以结合到每个合成的四路交界处，并提供证据，在高蛋白质：DNA比例的交界处结构的显着失真。Hjc晶体结构中的晶体堆积相互作用的分析表明了这种自抑制的分子基础。这些研究结果的DNA-蛋白质相互作用的定量研究的更广泛的影响进行了讨论。
The Holliday junction-resolving enzyme Me is conserved in the archaea and probably plays a role analogous to that of Escherichia coli RuvC in the pathway of homologous recombination. Me specifically recognizes four-way DNA junctions, cleaving them without sequence preference to generate recombinant DNA duplex products. Hjc imposes an X-shaped global conformation on the bound DNA junction and distorts base stacking around the point of cleavage, three nucleotides 3' of the junction center. We show that Me is autoinhibitory under single turnover assay conditions and that this can be relieved by the addition of either competitor duplex DNA or the architectural double-stranded DNA-binding protein Sso7d (i.e. by approximating in vivo conditions more closely). Using a combination of isothermal titration calorimetry and fluorescent resonance energy transfer, we demonstrate that multiple Hjc dimers can bind to each synthetic four-way junction and provide evidence for significant distortion of the junction structure at high protein:DNA ratios. Analysis of crystal packing interactions in the crystal structure of Hjc suggests a molecular basis for this autoinhibition. The wider implications of these findings for the quantitative study of DNA-protein interactions is discussed.