A Distinct Triplex DNA Unwinding Activity of ChlR1 Helicase
A Distinct Triplex DNA Unwinding Activity of ChlR1 Helicase
复制标题
DOI：
10.1074/jbc.m114.634923
复制
发表时间：
2015-02-20
影响因子：
4.8
通讯作者：
Wu, Yuliang
中科院分区：
文献类型：
--
作者：
Guo, Manhong;Hundseth, Kristian;Wu, Yuliang
人类ChlR1（DDX11）基因的突变与一种独特的遗传性疾病相关，称为华沙断裂综合征，其特征是基因组维护中的细胞缺陷。通过Hoogsteen或反向Hoogsteen氢键形成的DNA三链体螺旋结构是可能是基因组不稳定性来源的替代DNA结构的实例。在这项研究中，我们研究了人类ChlR1解旋酶的能力，使DNA三链体不稳定。生物化学研究表明，ChlR1有效地融化分子间和分子内的DNA三链体底物在ATP依赖性的方式。与其他底物如复制叉和G-四链体DNA相比，三链体DNA是ChlR1的首选底物。此外，与FANCJ，同一家族的解旋酶相比，ChlR1的三链体拆分活性是独特的。另一方面，来自华沙断裂综合征患者的突变蛋白未能解开这些三链体。先前表征的三链体DNA特异性抗体（Jel 466）结合三链体DNA结构并抑制ChlRl解旋活性。此外，细胞分析表明，有增加的三链DNA含量和双链断裂ChlR1耗尽的细胞，但不是在FANCJ（-/-）细胞，当细胞用三链稳定化合物苯并喹喔啉，这表明ChlR1三螺旋结构的熔化是独特的和生理上重要的，以捍卫基因组的完整性。根据我们的研究结果，我们得出结论，已知存在于体内的丰富的ChlR1可能是一个强大的威慑力的稳定性的三链体，可以潜在地形成在人类基因组中。
Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw breakage syndrome characterized by cellular defects in genome maintenance. The DNA triplex helix structures that form by Hoogsteen or reverse Hoogsteen hydrogen bonding are examples of alternate DNA structures that can be a source of genomic instability. In this study, we have examined the ability of human ChlR1 helicase to destabilize DNA triplexes. Biochemical studies demonstrated that ChlR1 efficiently melted both intermolecular and intramolecular DNA triplex substrates in an ATP-dependent manner. Compared with other substrates such as replication fork and G-quadruplex DNA, triplex DNA was a preferred substrate for ChlR1. Also, compared with FANCJ, a helicase of the same family, the triplex resolving activity of ChlR1 is unique. On the other hand, the mutant protein from a Warsaw breakage syndrome patient failed to unwind these triplexes. A previously characterized triplex DNA-specific antibody (Jel 466) bound triplex DNA structures and inhibited ChlR1 unwinding activity. Moreover, cellular assays demonstrated that there were increased triplex DNA content and double-stranded breaks in ChlR1-depleted cells, but not in FANCJ(-/-) cells, when cells were treated with a triplex stabilizing compound benzoquinoquinoxaline, suggesting that ChlR1 melting of triple-helix structures is distinctive and physiologically important to defend genome integrity. On the basis of our results, we conclude that the abundance of ChlR1 known to exist in vivo is likely to be a strong deterrent to the stability of triplexes that can potentially form in the human genome.