Hinde Elizabeth, Kong Xiangduo, Yokomori Kyoko, Gratton Enrico
Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, California; School of Medical Sciences and Australian Centre for NanoMedicine, University of New South Wales, Sydney, Australia.
Department of Biological Chemistry, School of Medicine, University of California, Irvine, California.
Biophys J. 2014 Jul 1;107(1):55-65. doi: 10.1016/j.bpj.2014.05.027.
Chromatin dynamics modulate DNA repair factor accessibility throughout the DNA damage response. The spatiotemporal scale upon which these dynamics occur render them invisible to live cell imaging. Here we present a believed novel assay to monitor the in vivo structural rearrangements of chromatin during DNA repair. By pair correlation analysis of EGFP molecular flow into chromatin before and after damage, this assay measures millisecond variations in chromatin compaction with submicron resolution. Combined with laser microirradiation we employ this assay to monitor the real-time accessibility of DNA at the damage site. We find from comparison of EGFP molecular flow with a molecule that has an affinity toward double-strand breaks (Ku-EGFP) that DNA damage induces a transient decrease in chromatin compaction at the damage site and an increase in compaction to adjacent regions, which together facilitate DNA repair factor recruitment to the lesion with high spatiotemporal control.
染色质动力学在整个DNA损伤反应过程中调节DNA修复因子的可及性。这些动力学发生的时空尺度使得它们在活细胞成像中不可见。在这里，我们提出了一种据信新颖的检测方法，用于监测DNA修复过程中染色质的体内结构重排。通过对损伤前后流入染色质的EGFP分子流进行配对相关分析，该检测方法以亚微米分辨率测量染色质压缩的毫秒级变化。结合激光微照射，我们使用该检测方法监测损伤部位DNA的实时可及性。通过将EGFP分子流与对双链断裂具有亲和力的分子（Ku-EGFP）进行比较，我们发现DNA损伤会导致损伤部位染色质压缩的短暂降低以及相邻区域压缩的增加，这两者共同促进DNA修复因子在高时空控制下募集到损伤部位。
