Mojumdar Aditya, De March Matteo, Marino Francesca, Onesti Silvia
From the Structural Biology Laboratory, Elettra-Sincrotrone Trieste, 34149 Basovizza, Trieste, Italy and.
the Dipartimento di Scienze della Vita, Università degli Studi di Trieste, 34127 Trieste, Italy.
J Biol Chem. 2017 Mar 10;292(10):4176-4184. doi: 10.1074/jbc.M116.767954. Epub 2016 Dec 20.
RecQ helicases are essential in the maintenance of genome stability. Five paralogues (RecQ1, Bloom, Werner, RecQ4, and RecQ5) are found in human cells, with distinct but overlapping roles. Mutations in human RecQ4 give rise to three distinct genetic disorders (Rothmund-Thomson, RAPADILINO, and Baller-Gerold syndromes), characterized by genetic instability, growth deficiency, and predisposition to cancer. Previous studies suggested that RecQ4 was unique because it did not seem to contain a RecQ C-terminal region (RQC) found in the other RecQ paralogues; such a region consists of a zinc domain and a winged helix domain and plays an important role in enzyme activity. However, our recent bioinformatic analysis identified in RecQ4 a putative RQC. To experimentally confirm this hypothesis, we report the purification and characterization of the catalytic core of human RecQ4. Inductively coupled plasma-atomic emission spectrometry detected the unusual presence of two zinc clusters within the zinc domain, consistent with the bioinformatic prediction. Analysis of site-directed mutants, targeting key RQC residues (putative zinc ligands and the aromatic residue predicted to be at the tip of the winged helix β-hairpin), showed a decrease in DNA binding, unwinding, and annealing, as expected for a functional RQC domain. Low resolution structural information obtained by small angle X-ray scattering data suggests that RecQ4 interacts with DNA in a manner similar to RecQ1, whereas the winged helix domain may assume alternative conformations, as seen in the bacterial enzymes. These combined results experimentally confirm the presence of a functional RQC domain in human RecQ4.
RecQ解旋酶对于维持基因组稳定性至关重要。在人类细胞中发现了五个同源物（RecQ1、布鲁姆综合征蛋白、沃纳综合征蛋白、RecQ4和RecQ5），它们具有不同但重叠的作用。人类RecQ4的突变会引发三种不同的遗传疾病（罗思蒙德 - 汤姆森综合征、RAPADILINO综合征和巴勒 - 杰罗尔德综合征），其特征为遗传不稳定、生长缺陷和易患癌症。先前的研究表明RecQ4是独特的，因为它似乎不包含在其他RecQ同源物中发现的RecQ C末端区域（RQC）；这样一个区域由一个锌结构域和一个翼状螺旋结构域组成，并且在酶活性中起重要作用。然而，我们最近的生物信息学分析在RecQ4中鉴定出一个推定的RQC。为了通过实验证实这一假设，我们报告了人类RecQ4催化核心的纯化和特性分析。电感耦合等离子体原子发射光谱法检测到锌结构域内异常存在两个锌簇，这与生物信息学预测一致。对靶向关键RQC残基（推定的锌配体和预计位于翼状螺旋β发夹末端的芳香族残基）的定点突变体的分析表明，如功能性RQC结构域所预期的那样，DNA结合、解旋和退火能力下降。通过小角X射线散射数据获得的低分辨率结构信息表明，RecQ4与DNA的相互作用方式类似于RecQ1，而翼状螺旋结构域可能呈现出替代构象，如在细菌酶中所见。这些综合结果通过实验证实了人类RecQ4中存在功能性RQC结构域。
