【摘要】 同源重组过程由重组酶介导,对维持细胞的遗传稳定性有极大的作用.链交换是同源重组的关键过程,研究链交换发生的基本步长对理解整个反应机制有着重要的作用. Rec A作为原核生物重组酶的重要成员,近年来持续受到广泛关注,但Rec A介导的同源重组链交换的步长目前还有争议.现在主流的观点认为链交换步长为3 bp,而我们的前期工作测得步长的最可几值为9 bp.为了进一步验证我们的结论,进而为更深层次的机理研究提供基础,本文采用酶切保护实验和单分子磁镊,配合使用不同的错配碱基序列从侧面验证了链交换步长不为3 bp,而更倾向于9 bp,并分析了一个步长内的错配碱基数目和分布对链交换进程的影响.该结论为进一步探索重组酶工作的分子机理提供了基础和新的思路.更多还原

【Abstract】 Homologous recombination(HR) which is important for maintaining the genetic stability of the cell is mainly mediated by recombinase. As a critical recombinase exists in prokaryotic organism, RecA has been widely studied. RecA could bind single strand DNA to form a filament structure to perform strand invasion and exchange with homologous DNA. The basic strand exchange step is a pathway to investigating the mechanism of strand exchange process. However, the specific step length is still controversial. The mainstream view is to regard the basic step as 3 bp, which sounds reasonable because the basic unit of RecA filament is a RecA monomer which binds 3 base pairs, but our previous study found that the most probable basic strand exchange step is 9 bp. In this research, we set up a series of experiments to demonstrate that the basic strand exchange step is 9 bp and how the mismatch base pairs in 9 bp affect the strand exchange process. We ues digestion protection to confirm whether the strand exchange process is completed and we employ a magnetic tweezer to measure how many base pairs are exchanged by strand exchange process. The digestion protection experiments show the strand exchange cannot overcome 33% mismatch sequence, but according to the result that strand exchange could overcome a single mismatch base pair, so that the step is not 3 bp. According to strand exchange length of 33% mismatch sequence, we find a 9 bp interval between the main peaks, which implies that the strand exchange step should be 9 bp rather than 3 bp. We also use continuous mismatch base pairs instead of periodic mismatch sequence to see whether the strand exchange process can be overcome. We find that the more the mismatch base pairs, the harder the strand exchange process passes across. Homology degree and its distribution both affect the strand exchange process. In general, the results suggest that the strand exchange step mediated by RecA is 9 bp, and the number of mismatched base pairs and their distribution influence the strand exchange process. The combination of digestion protection assay and magnetic tweezers can further take advantage of single molecule techniques to investigate mechanism of HR.更多还原