Department of Genetics, Erasmus University Medical Center, GE Rotterdam, The Netherlands.
DNA Repair (Amst). 2011 Jul 15;10(7):760-71. doi: 10.1016/j.dnarep.2011.04.015. Epub 2011 May 7.
Despite detailed knowledge on the genetic network and biochemical properties of most of the nucleotide excision repair (NER) proteins, cell biological analysis has only recently made it possible to investigate the temporal and spatial organization of NER. In contrast to several other DNA damage response mechanisms that occur in specific subnuclear structures, NER is not confined to nuclear foci, which has severely hampered the analysis of its arrangement in time and space. In this review the recently developed tools to study the dynamic molecular transactions between the NER factors and the chromatin template are summarized. First, different procedures to inflict DNA damage in a part of the cell nucleus are discussed. In addition, technologies to measure protein dynamics of NER factors tagged with the green fluorescent protein (GFP) will be reviewed. Most of the dynamic parameters of GFP-tagged NER factors are deduced from different variants of 'fluorescence recovery after photobleaching' (FRAP) experiments and FRAP analysis procedures will be briefly evaluated. The combination of local damage induction, genetic tagging of repair factors with GFP and microscopy innovations have provided the basis for the determination of NER kinetics within living mammalian cells. These new cell biological approaches have disclosed a highly dynamic arrangement of NER factors that assemble in an orderly fashion on damaged DNA. The spatio-temporal analysis tools developed for the study of NER and the kinetic model derived from these studies can serve as a paradigm for the understanding of other chromatin-associated processes.
尽管人们对大多数核苷酸切除修复 (NER) 蛋白的遗传网络和生化特性有详细的了解，但细胞生物学分析直到最近才能够研究 NER 的时空组织。与其他几种发生在特定核亚结构中的 DNA 损伤反应机制不同，NER 并不局限于核斑点，这严重阻碍了对其在时间和空间上排列的分析。在这篇综述中，总结了最近开发的用于研究 NER 因子与染色质模板之间动态分子相互作用的工具。首先，讨论了在细胞核的一部分中引起 DNA 损伤的不同程序。此外，还将回顾用于测量用绿色荧光蛋白 (GFP) 标记的 NER 因子蛋白动力学的技术。GFP 标记的 NER 因子的大多数动态参数都来自不同的“光漂白后荧光恢复”（FRAP）实验的变体，并且将简要评估 FRAP 分析程序。局部损伤诱导、用 GFP 遗传标记修复因子以及显微镜创新的结合为在活哺乳动物细胞中确定 NER 动力学奠定了基础。这些新的细胞生物学方法揭示了 NER 因子在受损 DNA 上以有序方式组装的高度动态排列。为研究 NER 而开发的时空分析工具以及从这些研究中得出的动力学模型，可以作为理解其他与染色质相关过程的范例。
