van der Heijden Thijn, van Noort John, van Leest Hendrikje, Kanaar Roland, Wyman Claire, Dekker Nynke H, Dekker Cees
Kavli Institute of Nanoscience, Delft University of Technology Lorentzweg 1, 2628 CJ Delft, The Netherlands.
Nucleic Acids Res. 2005 Apr 11;33(7):2099-105. doi: 10.1093/nar/gki512. Print 2005.
The assembly of RecA onto a torsionally constrained double-stranded DNA molecule was followed in real time using magnetic tweezers. Formation of a RecA-DNA filament on the DNA tether was stalled owing to different physical processes depending on the applied stretching force. For forces up to 3.6 pN, the reaction stalled owing to the formation of positive plectonemes in the remaining DNA molecule. Release of these plectonemes by rotation of the magnets led to full coverage of the DNA molecule by RecA. At stretching forces larger than 3.6 pN, the twist induced during filament formation caused the reaction to stall before positive supercoils were generated. We deduce a maximum built-up torsion of 10.1 +/- 0.7 k(b)T. In vivo this built-up torsion may be used to favor regression of a stalled replication fork or to free the chromosomal DNA in E.coli from its condensing proteins.
使用磁镊实时跟踪RecA在受扭转约束的双链DNA分子上的组装过程。根据所施加的拉伸力，由于不同的物理过程，DNA系链上RecA-DNA细丝的形成会受阻。对于高达3.6皮牛的力，反应受阻是由于剩余DNA分子中形成了正超螺旋。通过磁体旋转释放这些超螺旋会导致RecA完全覆盖DNA分子。在大于3.6皮牛的拉伸力下，细丝形成过程中诱导的扭曲会导致反应在产生正超螺旋之前就停滞。我们推断最大累积扭转力为10.1±0.7k(b)T。在体内，这种累积扭转力可用于促进停滞的复制叉的回归或使大肠杆菌中的染色体DNA从其凝聚蛋白中释放出来。
