Mutational phospho-mimicry reveals a regulatory role for the XRCC4 and XLF C-terminal tails in modulating DNA bridging during classical non-homologous end joining
Mutational phospho-mimicry reveals a regulatory role for the XRCC4 and XLF C-terminal tails in modulating DNA bridging during classical non-homologous end joining
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DOI：
10.7554/elife.22900
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发表时间：
2017-05-13
期刊：
影响因子：
7.7
通讯作者：
Modesti, Mauro
中科院分区：
文献类型：
--
作者：
Normanno, Davide;Negrel, Aurelie;Modesti, Mauro
XRCC4和DNA连接酶4 （LIG4）形成紧密复合物，为经典的非同源末端连接（高等真核生物中主要的DNA双链断裂修复途径）提供DNA连接酶活性，并由XLF刺激。独立于LIG4， XLF也与XRCC4结合形成连接DNA的细丝。这些XRCC4/XLF复合物快速加载和连接断裂的DNA，从而刺激分子间的连接。XRCC4和XLF都包含无序的c端尾，在分离时功能上是不可缺少的，但在DNA- pk和/或ATM的DNA损伤反应中被磷酸化。在这里，我们同时修饰XRCC4和XLF的尾部，用丙氨酸或天冬氨酸残基取代先前鉴定的14个磷酸化位点。这些磷酸化阻断和模仿突变会影响XRCC4/XLF复合物的稳定性和DNA桥接能力，但不会影响其刺激LIG4活性的能力。这一发现暗示磷酸化可能调节XRCC4/XLF丝的DNA桥接。
XRCC4 and DNA Ligase 4 (LIG4) form a tight complex that provides DNA ligase activity for classical non-homologous end joining (the predominant DNA double-strand break repair pathway in higher eukaryotes) and is stimulated by XLF. Independently of LIG4, XLF also associates with XRCC4 to form filaments that bridge DNA. These XRCC4/XLF complexes rapidly load and connect broken DNA, thereby stimulating intermolecular ligation. XRCC4 and XLF both include disordered C-terminal tails that are functionally dispensable in isolation but are phosphorylated in response to DNA damage by DNA-PK and/or ATM. Here we concomitantly modify the tails of XRCC4 and XLF by substituting fourteen previously identified phosphorylation sites with either alanine or aspartate residues. These phospho-blocking and-mimicking mutations impact both the stability and DNA bridging capacity of XRCC4/XLF complexes, but without affecting their ability to stimulate LIG4 activity. Implicit in this finding is that phosphorylation may regulate DNA bridging by XRCC4/XLF filaments.