Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520, USA.
DNA Repair (Amst). 2013 Sep;12(9):707-12. doi: 10.1016/j.dnarep.2013.05.004. Epub 2013 Jun 12.
The role of Dmc1 as a meiosis-specific general recombinase was first demonstrated in Saccharomyces cerevisiae. Progress in understanding the biochemical mechanism of ScDmc1 has been hampered by its tendency to form inactive aggregates. We have found that the inclusion of ATP during protein purification prevents Dmc1 aggregation. ScDmc1 so prepared is capable of forming D-loops and responsive to its accessory factors Rad54 and Rdh54. Negative staining electron microscopy and iterative helical real-space reconstruction revealed that the ScDmc1-ssDNA nucleoprotein filament harbors 6.5 protomers per turn with a pitch of ∼106Å. The ScDmc1 purification procedure and companion molecular analyses should facilitate future studies on this recombinase.
Dmc1 在减数分裂中作为一种通用重组酶的作用最初是在酿酒酵母中被证明的。尽管人们在理解 ScDmc1 的生化机制方面取得了一些进展，但由于其易于形成无活性的聚集体，这一进程受到了阻碍。我们发现，在蛋白质纯化过程中加入 ATP 可以防止 Dmc1 聚集。用这种方法制备的 ScDmc1 能够形成 D 环，并对其辅助因子 Rad54 和 Rdh54 作出反应。负染色电子显微镜和迭代螺旋实空间重建显示，ScDmc1-ssDNA 核蛋白丝每转含有 6.5 个亚基，螺距约为 106Å。ScDmc1 的纯化程序和伴随的分子分析应该有助于今后对这种重组酶的研究。
