Insight into the Roles of Helicase Motif Ia by Characterizing Fanconi Anemia Group J Protein ( FANCJ) Patient Mutations
Insight into the Roles of Helicase Motif Ia by Characterizing Fanconi Anemia Group J Protein ( FANCJ) Patient Mutations
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DOI：
10.1074/jbc.m113.538892
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发表时间：
2014-04-11
影响因子：
4.8
通讯作者：
Wu, Yuliang
中科院分区：
文献类型：
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作者：
Guo, Manhong;Vidhyasagar, Venkatasubramanian;Wu, Yuliang
背景：两个Fanconi贫血患者的错义突变位于FANCJ解旋酶基序Ia。结果：突变体R251 C使FANCJ的DNA结合能力降低，Q255 H使解旋酶活性与DNA转位不偶联。结论：解旋酶Ia基序在FANCJ酶活性和DNA修复中起重要作用。重要性：解旋酶模体Ia不仅参与核酸结合，还参与ATP结合和ATP水解与解旋的偶联，解旋酶是将ATP水解能量与结构化DNA或RNA的解旋和重构偶联的分子马达，解旋酶模体Ia与保守的解旋酶模体相互协调。FANCJ是一种DNA解旋酶，与范可尼贫血、乳腺癌和卵巢癌有遗传联系。在这里，我们的特点是两个范可尼贫血患者的突变，R251 C和Q255 H，这是本地化的解旋酶基序Ia。我们的遗传互补分析表明，R251 C和Q255 H等位基因都未能挽救顺铂敏感性的FANCJ空细胞系检测细胞存活或-H2 AX灶形成。此外，我们的生化分析表明，这两种纯化的重组蛋白废除DNA解旋酶活性，并未能破坏DNA-蛋白质复合物。有趣的是，与野生型FANCJ蛋白相比，R251 C损害了DNA与单链DNA和双链DNA的结合能力，而Q255 H保留了与这些DNA底物更高的结合活性。因此，R251 C取消了其依赖DNA的ATP水解活性，而Q255 H保留了正常的ATP酶活性。物理上，R251 C的ATP结合能力降低，而Q255 H的ATP结合能力正常，并能在单链DNA上转位。尽管这两种蛋白质在我们的激光激活共聚焦测定中都被招募到损伤位点，但它们失去了DNA修复功能，这解释了为什么它们在野生型背景中表达时会产生结构域负效应。总之，我们的工作不仅揭示了解旋酶基序Ia的结构功能，而且还提供了FANCJ在相关疾病中的分子病理学。
Background: Two Fanconi anemia patient missense mutations are localized in FANCJ helicase motif Ia. Results: Mutant R251C impairs the DNA binding ability of FANCJ; Q255H uncouples DNA translocation from helicase activity. Conclusion: Helicase motif Ia plays critical roles in FANCJ enzymatic activity and DNA repair. Significance: Helicase motif Ia is involved not only in nucleic acid binding but also ATP binding and coupling ATP hydrolysis to unwinding.Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding and remodeling of structured DNA or RNA, which is coordinated by conserved helicase motifs. FANCJ is a DNA helicase that is genetically linked to Fanconi anemia, breast cancer, and ovarian cancer. Here, we characterized two Fanconi anemia patient mutations, R251C and Q255H, that are localized in helicase motif Ia. Our genetic complementation analysis revealed that both the R251C and Q255H alleles failed to rescue cisplatin sensitivity of a FANCJ null cell line as detected by cell survival or -H2AX foci formation. Furthermore, our biochemical assays demonstrated that both purified recombinant proteins abolished DNA helicase activity and failed to disrupt the DNA-protein complex. Intriguingly, R251C impaired DNA binding ability to single-strand DNA and double-strand DNA, whereas Q255H retained higher binding activity to these DNA substrates compared with wild-type FANCJ protein. Consequently, R251C abolished its DNA-dependent ATP hydrolysis activity, whereas Q255H retained normal ATPase activity. Physically, R251C had reduced ATP binding ability, whereas Q255H had normal ATP binding ability and could translocate on single-strand DNA. Although both proteins were recruited to damage sites in our laser-activated confocal assays, they lost their DNA repair function, which explains why they exerted a domain negative effect when expressed in a wild-type background. Taken together, our work not only reveals the structural function of helicase motif Ia but also provides the molecular pathology of FANCJ in related diseases.