Hazel Raphael D, Tian Kegui, de Los Santos Carlos
Department of Physiology & Biophysics, and Department of Pharmacological Sciences, Stony Brook University, School of Medicine, Stony Brook, New York 11794-8651, USA.
Biochemistry. 2008 Nov 18;47(46):11909-19. doi: 10.1021/bi800950t. Epub 2008 Oct 25.
Ionizing radiation produces clustered lesions in DNA. Since the orientation of bistranded lesions affects their recognition by DNA repair enzymes, clustered damages are more difficult to process and thus more toxic than single oxidative lesions. In order to understand the structural determinants that lead to differential recognition, we used NMR spectroscopy and restrained molecular dynamics to solve the structure of two DNA duplexes, each containing two stable abasic site analogues positioned on opposite strands of the duplex and staggered in the 3' (-1 duplex, (AP) 2-1 duplex) or 5' (+1 duplex, (AP) 2+1 duplex) direction. Cross-peak connectivities observed in the nonexchangeable NOESY spectra indicate compression of the helix at the lesion site of the duplexes, resulting in the formation of two abasic bulges. The exchangeable proton spectra show the AP site partner nucleotides forming interstrand hydrogen bonds that are characteristic of a Watson-Crick G.C base pairs, confirming the extra helical nature of the AP residues. Restrained molecular dynamics simulations generate a set of converging structures in full agreement with the spectroscopic data. In the (AP) 2-1 duplex, the extra helical abasic site residues reside in the minor groove of the helix, while they appear in the major groove in the (AP) 2+1 duplex. These structural differences are consistent with the differential recognition of bistranded abasic site lesions by human AP endonuclease.
电离辐射会在DNA中产生簇状损伤。由于双链损伤的方向会影响DNA修复酶对它们的识别，所以簇状损伤比单个氧化损伤更难处理，因此毒性也更大。为了了解导致差异识别的结构决定因素，我们使用核磁共振光谱法和受限分子动力学来解析两个DNA双链体的结构，每个双链体都包含两个稳定的无碱基位点类似物，它们位于双链体的相反链上，并在3'（-1双链体，(AP)2-1双链体）或5'（+1双链体，(AP)2+1双链体）方向上交错排列。在不可交换的核Overhauser效应光谱（NOESY）中观察到的交叉峰连接性表明，双链体损伤位点处的螺旋发生了压缩，导致形成了两个无碱基凸起。可交换质子光谱显示，无碱基位点的配对核苷酸形成了链间氢键，这是沃森-克里克G.C碱基对的特征，证实了无碱基残基的螺旋外性质。受限分子动力学模拟生成了一组与光谱数据完全一致的收敛结构。在(AP)2-1双链体中，螺旋外的无碱基位点残基位于螺旋的小沟中，而在(AP)2+1双链体中，它们出现在大沟中。这些结构差异与人类无碱基内切核酸酶对双链无碱基位点损伤的差异识别是一致的。
