Rothenberg Eli, Grimme Jill M, Spies Maria, Ha Taekjip
Howard Hughes Medical Institute and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Proc Natl Acad Sci U S A. 2008 Dec 23;105(51):20274-9. doi: 10.1073/pnas.0810317106. Epub 2008 Dec 11.
The Rad52 protein has critical functions in distinct pathways of the homology-directed DNA repair, one of which is to promote the annealing of complementary strands of DNA. Both yeast and human Rad52 proteins organize into ring-shaped oligomers with the predominant form being a heptamer. Despite the wealth of information obtained in previous investigations, how Rad52 mediates homology search and annealing remains unclear. Here, we developed single-molecule fluorescence resonance energy transfer approaches to probe hRad52-mediated DNA annealing events in real time. We found that annealing proceeds in successive steps involving rearrangements of the ssDNA-hRad52 complex. Moreover, after initial pairing, further search for extended homology occurs without dissociation. This search process is driven by an interaction between 2 overlapping nucleoprotein complexes. In light of these observations we propose a model for hRad52-mediated DNA annealing where ssDNA release and dsDNA zippering are coordinated through successive rearrangement of overlapping nucleoprotein complexes.
Rad52蛋白在同源性指导的DNA修复的不同途径中具有关键功能，其中之一是促进DNA互补链的退火。酵母和人类的Rad52蛋白都组装成环形寡聚体，主要形式是七聚体。尽管先前的研究获得了大量信息，但Rad52如何介导同源性搜索和退火仍不清楚。在这里，我们开发了单分子荧光共振能量转移方法来实时探测hRad52介导的DNA退火事件。我们发现退火过程是连续进行的，涉及单链DNA-hRad52复合物的重排。此外，在初始配对后，无需解离即可进一步搜索更长的同源序列。这个搜索过程是由两个重叠的核蛋白复合物之间的相互作用驱动的。基于这些观察结果，我们提出了一个hRad52介导的DNA退火模型，其中单链DNA的释放和双链DNA的拉链化是通过重叠核蛋白复合物的连续重排来协调的。
