Grigoriev M, Hsieh P
Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-1810, USA.
Mol Cell Biol. 1997 Dec;17(12):7139-50. doi: 10.1128/MCB.17.12.7139.
The Holliday junction is a key intermediate in genetic recombination. Here, we examine the effect of a nucleosome core on movement of the Holliday junction in vitro by spontaneous branch migration. Histone octamers consisting of H2A, H2B, H3, and H4 are reconstituted onto DNA duplexes containing an artificial nucleosome-positioning sequence consisting of a tandem array of an alternating AT-GC sequence motif. Characterization of the reconstituted branch migration substrates by micrococcal nuclease mapping and exonuclease III and hydroxyl radical footprinting reveal that 70% of the reconstituted octamers are positioned near the center of the substrate and the remaining 30% are located at the distal end, although in both cases some translational degeneracy is observed. Branch migration assays with the octamer-containing substrates reveal that the Holliday junction cannot migrate spontaneously through DNA organized into a nucleosomal core unless DNA-histone interactions are completely disrupted. Similar results are obtained with branch migration substrates containing an octamer positioned on a naturally occurring sequence derived from the yeast GLN3 locus. Digestion of Holliday junctions with T7 endonuclease I establishes that the junction is not trapped by the octamer but can branch migrate in regions free of histone octamers. Our findings suggest that migration of Holliday junctions during recombination and the recombinational repair of DNA damage requires proteins not only to accelerate the intrinsic rate of branch migration but also to facilitate the passage of the Holliday junction through a nucleosome.
霍利迪连接体是基因重组中的关键中间体。在此，我们通过自发分支迁移研究了核小体核心对体外霍利迪连接体移动的影响。由H2A、H2B、H3和H4组成的组蛋白八聚体被重组到含有由交替的AT - GC序列基序串联阵列组成的人工核小体定位序列的DNA双链体上。通过微球菌核酸酶图谱分析、核酸外切酶III和羟基自由基足迹分析对重组后的分支迁移底物进行表征，结果显示70%的重组八聚体位于底物中心附近，其余30%位于远端，不过在这两种情况下都观察到了一些平移简并性。对含八聚体底物进行的分支迁移实验表明，除非DNA - 组蛋白相互作用被完全破坏，否则霍利迪连接体无法自发穿过组装成核小体核心的DNA。对于含有位于源自酵母GLN3基因座的天然序列上的八聚体的分支迁移底物，也获得了类似结果。用T7内切核酸酶I消化霍利迪连接体表明，连接体不会被八聚体捕获，但可以在不含组蛋白八聚体的区域进行分支迁移。我们的研究结果表明，在重组过程中霍利迪连接体的迁移以及DNA损伤的重组修复不仅需要蛋白质来加速分支迁移的内在速率，还需要蛋白质来促进霍利迪连接体穿过核小体。
