Lee Andrew J, Sharma Rajan, Hobbs Jamie K, Wälti Christoph
Bioelectronics Group, School of Electronic & Electrical Engineering, University of Leeds, Woodhouse Lane, Leeds, LS2 9JT, UK.
Department of Physics and Astronomy, University of Sheffield, Hounsfield Road, Sheffield, S3 7RH, UK.
Nucleic Acids Res. 2017 Nov 16;45(20):11743-11751. doi: 10.1093/nar/gkx769.
The mechanism by which pre-synaptic RecA nucleoprotein filaments efficiently locate sequence homology across genomic DNA remains unclear. Here, using atomic force microscopy, we directly investigate the intermediates of the RecA-mediated homologous recombination process and find it to be highly cooperative, involving multiple phases. Initially, the process is dominated by a rapid 'association' phase, where multiple filaments interact on the same dsDNA simultaneously. This cooperative nature is reconciled by the observation of localized dense clusters of pre-synaptic filaments interacting with the observed dsDNA molecules. This confinement of reactive species within the vicinity of the dsDNA, is likely to play an important role in ensuring that a high interaction rate between the nucleoprotein filaments and the dsDNA can be achieved. This is followed by a slower 'resolution' phase, where the synaptic joints either locate sequence homology and progress to a post-synaptic joint, or dissociate from the dsDNA. Surprisingly, the number of simultaneous synaptic joints decreases rapidly after saturation of the dsDNA population, suggesting a reduction in interaction activity of the RecA filaments. We find that the time-scale of this decay is in line with the time-scale of the dispersion of the RecA filament clusters, further emphasising the important role this cooperative phenomena may play in the RecA-facilitated homology search.
突触前RecA核蛋白细丝在基因组DNA上高效定位序列同源性的机制仍不清楚。在这里，我们使用原子力显微镜直接研究RecA介导的同源重组过程的中间体，发现它具有高度协同性，涉及多个阶段。最初，该过程以快速的“结合”阶段为主，多个细丝同时在同一条双链DNA上相互作用。突触前细丝与观察到的双链DNA分子相互作用形成局部密集簇，这一现象解释了这种协同性质。将反应性物种限制在双链DNA附近，可能在确保核蛋白细丝与双链DNA之间实现高相互作用率方面发挥重要作用。随后是较慢的“解离”阶段，此时突触接头要么定位序列同源性并进展为突触后接头，要么与双链DNA解离。令人惊讶的是，双链DNA群体饱和后，同时存在的突触接头数量迅速减少，这表明RecA细丝的相互作用活性降低。我们发现这种衰减的时间尺度与RecA细丝簇分散的时间尺度一致，进一步强调了这种协同现象在RecA促进的同源性搜索中可能发挥的重要作用。
