Castaño Alejandra, Roy Upasana, Schärer Orlando D
Stony Brook University, Stony Brook, NY, United States.
Stony Brook University, Stony Brook, NY, United States; Center for Genomic Integrity, Institute for Basic Science, Ulsan, Korea; Ulsan National Institute of Science and Technology, Ulsan, Korea.
Methods Enzymol. 2017;591:415-431. doi: 10.1016/bs.mie.2017.03.007. Epub 2017 Apr 14.
Nitrogen mustards (NMs) react with two bases on opposite strands of a DNA duplex to form a covalent linkage, yielding adducts called DNA interstrand cross-links (ICLs). This prevents helix unwinding, blocking essential processes such as replication and transcription. Accumulation of ICLs causes cell death in rapidly dividing cells, especially cancer cells, making ICL-forming agents like NMs valuable in chemotherapy. However, the repair of ICLs can contribute to chemoresistance through a number of pathways that remain poorly understood. One of the impediments in studying NM ICL repair mechanisms has been the difficulty of generating site-specific and stable NM ICLs. Here, we describe two methods to synthesize stable NM ICL analogs that make it possible to study DNA ICL repair. As a proof of principle of the suitability of these NM ICLs for biochemical and cell biological studies, we use them in primer extension assays with Klenow polymerase. We show that the NM ICL analogs block the polymerase activity and remain intact under our experimental conditions.
氮芥（NMs）与DNA双链体相反链上的两个碱基发生反应，形成共价连接，产生称为DNA链间交联（ICLs）的加合物。这会阻止螺旋解旋，阻碍诸如复制和转录等基本过程。ICLs的积累会导致快速分裂的细胞尤其是癌细胞死亡，这使得像NMs这样的ICL形成剂在化疗中具有重要价值。然而，ICLs的修复可通过一些仍知之甚少的途径导致化疗耐药。研究NM ICL修复机制的障碍之一是难以生成位点特异性且稳定的NM ICLs。在此，我们描述了两种合成稳定的NM ICL类似物的方法，这使得研究DNA ICL修复成为可能。作为这些NM ICLs适用于生化和细胞生物学研究的原理证明，我们将它们用于用klenow聚合酶进行的引物延伸试验中。我们表明，NM ICL类似物会阻断聚合酶活性，并且在我们的实验条件下保持完整。
