Characterization of DNA binding and pairing activities associated with the native SFPQ·NONO DNA repair protein complex.
Characterization of DNA binding and pairing activities associated with the native SFPQ·NONO DNA repair protein complex.
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DOI：
10.1016/j.bbrc.2015.05.024
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发表时间：
2015-08-07
影响因子：
3.1
通讯作者：
Dynan, William S.
中科院分区：
文献类型：
--
作者：
Udayakumar, Durga;Dynan, William S.
非同源末端连接(NHEJ)是DNA双链断裂修复的主要途径。我们以前已经证明，在NHEJ的早期阶段，SFPQ(PSF)和NONO(P54nrb)的复合体与Ku蛋白合作，形成一个固定的预连接复合体，并刺激末端连接活性10倍或更多。SFPQ和NONO与其他修复因子没有相似之处，其作用机制尚不清楚。在这里，我们使用一种优化的微孔法来表征从人(HeLa)细胞中纯化的天然SFPQ·NONO复合体的体外DNA结合行为。SFPQ·NONO和Ku蛋白独立地与DNA结合，几乎没有协同作用的证据，在高浓度时只有轻微的相互干扰。Ku蛋白需要游离的DNA末端才能结合，而SFPQ·NONO则不需要。Ku和SFPQ·NONO都具有配对活性，通过微孔分析中DNA结合蛋白捕获第二个DNA片段的能力来衡量。此外，SFPQ·NONO刺激DNA依赖的蛋白激酶自动磷酸化，这与在不阻断DNA末端的情况下促进突触复合体的形成的能力一致。这些发现表明，SFPQ·NONO通过与内部DNA序列结合并与其他修复蛋白合作稳定突触预连接复合体来促进末端连接。
Nonhomologous end joining (NHEJ) is a major pathway for repair of DNA double-strand breaks. We have previously shown that a complex of SFPQ (PSF) and NONO (p54nrb) cooperates with Ku protein at an early step of NHEJ, forming a committed preligation complex and stimulating end-joining activity by 10-fold or more. SFPQ and NONO show no resemblance to other repair factors, and their mechanism of action is uncertain. Here, we use an optimized microwell-based assay to characterize the in vitro DNA binding behavior of the native SFPQ•NONO complex purified from human (HeLa) cells. SFPQ•NONO and Ku protein bind independently to DNA, with little evidence of cooperativity and only slight mutual interference at high concentration. Whereas Ku protein requires free DNA ends for binding, SFPQ•NONO does not. Both Ku and SFPQ•NONO have pairing activity, as measured by the ability of DNA-bound protein to capture a second DNA fragment in a microwell-based assay. Additionally, SFPQ•NONO stimulates DNA-dependent protein kinase autophosphorylation, consistent with the ability to promote formation of a synaptic complex formation without occluding the DNA termini proper. These findings suggest that SFPQ•NONO promotes end joining by binding to internal DNA sequences and cooperating with other repair proteins to stabilize a synaptic pre-ligation complex.
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影响因子：
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