Mechanistic insight into the assembly of the HerA-NurA helicase-nuclease DNA end resection complex.
Mechanistic insight into the assembly of the HerA-NurA helicase-nuclease DNA end resection complex.
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DOI：
10.1093/nar/gkx890
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发表时间：
2017-11-16
影响因子：
14.9
通讯作者：
Politis A
中科院分区：
文献类型：
--
作者：
Ahdash Z;Lau AM;Byrne RT;Lammens K;Stüetzer A;Urlaub H;Booth PJ;Reading E;Hopfner KP;Politis A
HerA-NurA解旋酶-核酸酶复合物与Mre 11和Rad 50合作以协调双链DNA断裂的修复。然而，关于HerA-NurA的组装机制和激活知之甚少。通过将混合质谱与cryo-EM、计算和生物化学数据相结合，我们研究了HerA的寡聚体形成，并详细说明了嗜热古菌中HerA-NurA复合物与核苷酸结合的机制。我们发现，无ATP的HerA和HerA-DNA复合物主要存在于溶液中作为一个七聚体，并作为DNA加载中间体。NurA或ATP的结合稳定了六聚体HerA，表明HerA-NurA被底物和复合物组装激活。为了研究ATP在DNA易位和加工中的作用，我们研究了核苷酸如何与HerA-NurA相互作用。我们发现，虽然六聚体HerA以“全或无”的方式结合六个核苷酸，HerA-NurA具有高度协调的成对结合机制，并使双链DNA的易位和加工成为可能。利用分子动力学模拟，我们揭示了新的外部ATP和内部DNA结合位点之间的残基间的相互作用。总的来说，在这里，我们提出了一个逐步组装机制，详细说明了协同激活的HerA-NurA的ATP，这使得双链DNA的有效处理。
The HerA–NurA helicase–nuclease complex cooperates with Mre11 and Rad50 to coordinate the repair of double-stranded DNA breaks. Little is known, however, about the assembly mechanism and activation of the HerA–NurA. By combining hybrid mass spectrometry with cryo-EM, computational and biochemical data, we investigate the oligomeric formation of HerA and detail the mechanism of nucleotide binding to the HerA–NurA complex from thermophilic archaea. We reveal that ATP-free HerA and HerA-DNA complexes predominantly exist in solution as a heptamer and act as a DNA loading intermediate. The binding of either NurA or ATP stabilizes the hexameric HerA, indicating that HerA–NurA is activated by substrates and complex assembly. To examine the role of ATP in DNA translocation and processing, we investigated how nucleotides interact with the HerA–NurA. We show that while the hexameric HerA binds six nucleotides in an ‘all-or-none’ fashion, HerA–NurA harbors a highly coordinated pairwise binding mechanism and enables the translocation and processing of double-stranded DNA. Using molecular dynamics simulations, we reveal novel inter-residue interactions between the external ATP and the internal DNA binding sites. Overall, here we propose a stepwise assembly mechanism detailing the synergistic activation of HerA–NurA by ATP, which allows efficient processing of double-stranded DNA.