Analysis of vaccinia topoisomerase peptide inhibitors

拓扑异构酶控制DNA的超螺旋状态，参与DNA复制和转录等生物过程。痘苗病毒拓扑异构酶(VT)是一种与人类拓扑异构酶I(抗癌靶标)密切相关的典型Ib型酶。VT活性的特异性多肽抑制剂将被分离出来。这些抑制剂将有助于理解拓扑异构酶抑制的催化机制和性质。这些抑制肽将通过生化方法鉴定其抑制VT结合、切割和DNA底物宗教化的能力，并使用体内模型测试抗病毒活性，靶向VT。将使用突变的VT酶(具有明确的点突变)进一步表征抑制剂，突出受抑制肽影响的酶活性所涉及的残基。此外，与多肽相互作用的VT区域将通过多肽-酶交联来识别。最后，将通过DNA足迹来鉴定导致DNA构象变化的抑制效应。

Topoisomerase enzymes control the supercoiled state of DNA and are involved in biological processes such as DNA replication and transcription. Vaccinia virus topoisomerase (VT) is a protypical type Ib enzyme closely related to human topoisomerase I (anti-cancer target). Specific peptide inhibitors of VT activity will be isolated. These inhibitors will aid in understanding the catalytic mechanism and the nature of topoisomerase inhibition. The inhibitor peptides will be identified biochemically for their ability to inhibit VT binding, cleavage and religation of their DNA substrate and tested for anti-viral activity, targeting VT using an in vivo model. Inhibitors will be further characterized using mutant VT enzymes (with defined point mutations), highlighting residues involved in enzyme activity that are affected by the inhibitory peptide. Furthermore, regions of VT that interact with the peptides will be identified by peptide-enzyme crosslinking. Finally, the inhibitory effects resulting in DNA conformational changes will be identified by DNA footprinting.