Identification of post translational modifications of human Topoiosmerase 1 in the presence of camptothecin and CHK1 inhibitor SCH900776.

Topisomerase 1 (TOP1), an essential enzyme in humans, plays a critical role in relaxation of DNA supercoils during replication and transcription. The TOP1 reaction cycle involves an obligate TOP1-DNA covalent complex intermediate (TOP1cc), which is stabilized by TOP1 poisons like camptothecin. While cellular responses to potentially genotoxic TOP1ccs are widely studied, the regulation of TOP1 catalytic cycle itself inside the cell in poorly understood. In the current study, we have demonstrated a regulatory role of the master checkpoint kinase CHK1 in regulating the TOP1 catalytic cycle inside cells. We performed an in silico prediction of CHK1 cognate sites on TOP1. In order to validate the same, we isolated catalytically engaged TOP1 from human genomic DNA, and performed mass spectrometric analysis. Our analysis revealed one previously unreported putative CHK1 cognate site to be phosphorylated on TOP1 inside cells. We further employed site directed mutagenesis, molecular and cellular assays to establish the role played by CHK1 in regulating TOP1 catalysis.

拓扑异构酶1（Topoisomerase 1, TOP1）是人体必需的酶类，在DNA复制与转录过程中对DNA超螺旋的松弛发挥关键作用。TOP1的催化循环包含一个必需的TOP1-DNA共价复合物中间体（TOP1cc），此类中间体可被喜树碱（camptothecin）这类TOP1毒剂所稳定。尽管学界已对潜在遗传毒性TOP1cc的细胞应答开展了广泛研究，但细胞内TOP1催化循环自身的调控机制仍未被充分阐明。本研究证实了核心检验点激酶CHK1对细胞内TOP1催化循环的调控作用。我们通过in silico预测了TOP1上的CHK1同源底物位点；为验证上述预测结果，我们从人类基因组DNA中分离得到处于催化结合状态的TOP1，并开展了质谱分析。分析结果显示，细胞内TOP1上存在一处此前未被报道的、可被磷酸化的CHK1潜在底物位点。我们进一步通过定点诱变、分子与细胞实验，证实了CHK1对TOP1催化过程的调控作用。