A histone octamer blocks branch migration of a Holliday junction
A histone octamer blocks branch migration of a Holliday junction
复制标题
DOI：
10.1128/mcb.17.12.7139
复制
发表时间：
1997-12-01
影响因子：
5.3
通讯作者：
Hsieh, P
中科院分区：
文献类型：
--
作者：
Grigoriev, M;Hsieh, P
Holliday连接是基因重组的关键中间环节，在这里，我们通过自发的分支迁移来研究核小体核心在体外对Holliday连接运动的影响。由H_2A、H_2B、H_3和H_4组成的组蛋白八聚体被重组到含有人工核小体定位序列的DNA双链上，该序列由交替的AT-GC序列基序组成。用微球菌核酸酶作图、核酸外切酶III和羟基自由基足迹对重组的分支迁移底物进行表征表明，70%的重组八聚体位于底物中心附近，其余30%位于远端，尽管在这两种情况下都观察到了一些翻译退化。含八聚体底物的分支迁移分析表明，除非DNA-组蛋白相互作用被完全破坏，否则Holliday连接不能通过组成核小体核心的DNA自发迁移，使用包含八聚体的分支迁移底物获得了类似的结果，T7内切酶I消化Holliday连接建立了连接不被八聚体捕获，但可以在没有组蛋白八聚体的区域迁移，我们的发现表明，在重组和DNA损伤的重组修复过程中，Holliday连接的迁移不仅需要蛋白质加快分支迁移的固有速度，还需要促进Holliday连接通过核小体。
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.