Dynamics of mammalian NER proteins

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.