DNA同源重组的物理过程和同源识别的分子机制研究

Homologous recombination is a kind of genetic recombinations，which happens between DNA homologous sequences. It is very important to organism surviving and developing. RecA and Rad51 are recombinases from prokaryotes and eukaryotes respectively. They are highly conservative and their nature and functions are very similar. People have a general understanding of the basic process of homologous recombination mediated by RecA and Rad51, but many physical details and molecular mechanisms are still not clear. Homology recognition and strand exchange are two critical steps in the process of homologous recombination. However, they have not been directly observed so far due to the highly dynamic property of these two steps, and too small the deformation degree of the intermediate. It became a famous difficulty to biologists for many years. Using strategically experimental design to bypass the problem of time resolution and spatial resolution, we will research the processes of homology recognition and strand exchange mediated by RecA and Rad51 with longitudinal magnetic tweezers and single molecular fluorescence resonance energy transfer. We will carry out the real-time observation of homology recognition and strand exchange processes in single molecular level, and conclude the influences of the mismatch on homology recognition and strand exchange. Then illuminate the processes of homology recognition and strand exchange mediated by RecA and Rad51 from single molecular level, and explain the mechanisms of them.

同源重组是生物体内常见的发生在DNA同源序列之间的重组，它对产生生物多样性和维持遗传信息的稳定至关重要。RecA和Rad51是分别存在于原核和真核生物中的重组酶，其序列具有高度保守性，其性质和功能也非常相似。人们已大体了解了RecA和Rad51介导的同源重组的基本过程，但仍有很多物理细节和分子机理尚不清楚。同源识别和链交换是同源重组过程中最关键的两个步骤，然而由于这两个步骤具有高度动态的特性，且中间过程形变量极小，至今尚无手段直接观察到，成为困扰生物学家多年的难题。我们将利用巧妙的实验设计，绕开时间分辨率和空间分辨率的问题，用纵向磁镊和单分子荧光共振能量转移技术来对同源识别和链交换过程进行单分子水平的实时观察，并且研究当参与同源重组的DNA间存在一定比例错配时对同源识别和链交换产生的影响，最终从分子水平阐明RecA和Rad51介导的同源识别和链交换过程，并对其机理作出解释。

同源重组是生物体内常见的发生在DNA同源序列之间的重组，它对产生生物多样性和维持遗传信息的稳定至关重要。RecA是存在于原核生物中的重组酶，其序列具有高度保守性，其性质和功能在RecA蛋白家族中具有代表性。人们已大体了解了RecA介导的同源重组的基本过程，但仍有很多物理细节和分子机理尚不清楚。同源识别和链交换是同源重组过程中最关键的两个步骤，然而由于这两个步骤具有高度动态的特性，且中间过程形变量极小，至今尚无手段直接观察到，成为困扰生物学家多年的难题。我们利用单分子磁镊技术和单分子FRET技术，通过巧妙的实验设计，捕捉到了链交换的中间过程。并且在实验过程中，我们发现链交换的的终止位置可以间接反映链交换的中间过程动态细节。我们充分利用该信息，设计了一系列单分子实验，发现当存在错配序列时，链交换的会在错配序列之前提前终止。我们构建了一个链交换的动态模型，认为序列比对已经到达错配序列，而链交换未到达错配序列，中间的部分可能为过渡区域。已换链区、过渡区和试探区，这三者如同行波向前传递，当遇到错配序列时，试探区停在错配区，而换链区和过渡区停在匹配部分，导致换链终点离错配区还有一定的距离。我们通过单分子实验，捕捉到了链交换的中间态，并且详细推理了同源识别和链交换的动态过程。