對健全的基因體來說，DNA雙股斷裂(DNA double-strand break, DSB)是最危險的傷害。DSB修復失敗可造成基因體之不穩定與癌症形成。從酵母菌(Saccharomyces cerevisiae)至人類，DSB修復之機制極為保守。於酵母菌中，組蛋白修飾複合體NuA4聚集至DSB處以進行組蛋白H2A與H4之乙醯化，此舉被認作為鬆開染色質並使修復蛋白得以進入。NuA4之兩個單元蛋白，Eaf3與Yng2於試管實驗(in vitro)中分別能夠藉由chromo結構域以及PHD (plant homeodomain)結構域與組蛋白H3的第36個離胺酸(lysine)之兩個甲基化 (H3K36me2)以及第4個離胺酸之三個甲基化(H3K4me3)反應。於此研究中，我們發現突變Eaf3之chromo結構域(eaf3-chromo)或Yng2之PHD結構域(yng2-PHD)對於細胞生長及DNA修復皆無顯著影響。然而，結合chromo結構域與PHD結構域兩者之突變(eaf3-chromo yng2-PHD)卻戲劇性地出現對細胞生長及DNA修復方面的缺陷。除此以外，染色質免疫沉澱(chromatin immunoprecipitation, ChIP)實驗透露出大量的組蛋白H2A的第129個絲胺酸(serine)之磷酸化(H2AS129p)、組蛋白H4的第12個離胺酸之乙醯化(H4K12ac)及H3K36me2於DSB處累積。Yng2於DSB處大量累積，表示NuA4有效地聚集至DSB處。相形之下，eaf3-chromo yng2-PHD雙突變株之H4K12ac與H3K36me2戲劇性地下降，並且H2AS129p之活化延長，表示雙突變株損傷了NuA4之聚集與修復效率。組蛋白H3的K4之突變(H3K4R)阻斷此位置甲基化，也造成Yng2與H4K12ac戲劇性地下降，以及H2AS129p之活化延長，表示H3K4R突變也損傷了NuA4之聚集與修復效率。我們的結果顯示DSB能夠引發H2AS129p與H3K36me2於斷裂處的專一性修飾，藉由這些修飾過之組蛋白與NuA4之單元蛋白的交互作用，NuA4複合體方能依次地聚集。這些交互作用存在H2AS129p與Arp4、H3K36me2與chromo結構域以及H3K4me3與PHD結構域之間。這些多重的交互作用對於NuA4在DSB處之聚集，扮演鞏固強化之角色。

DNA double strand breaks (DSBs) are the most dangerous lesions to the integrity of genome. Failure to repair DSBs properly can lead to genomic instability and cancer. The mechanism of DSB repair is highly conserved from yeast to human. In yeast Saccharomyces cerevisiae, the histone modification complex, NuA4 (functional homolog of Tip60 in human) is recruited to DSBs where it acetylates histone H2A and H4, presumably relaxing the chromatin and allowing the access of repairing proteins. Two subunits of NuA4, Yng2 and Eaf3, can interact with methylated K4 and K36 of histone H3 (H3K4me and H3K36me) by their plant homeodomain (PHD) and chromo domain respectively in vitro. Here, we demonstrated that mutations either in the Yng2 PHD domain (yng2-PHD) or the Eaf3 chromo domain (eaf3-W84A/W88A) have no significant effect on cell growth or DNA repair. However, combined mutations both in the PHD and chromo domain (yng2-PHD eaf3-W84A/W88A) show dramatic defect both in cell growth and DNA damage repair. In addition, the chromatin immunoprecipitation (ChIP) experiments reveal that high level of phospho-S129 of histone H2A (H2AS129p), acetyl-K12 of histone H4 (H4K12ac), H3K4me2, H3K4me3, H3K36me2, and Yng2 are enriched at HO DSB site, suggesting histone H2A-S129p, H3K4me2, H3K4me3, and H3K36me2 are specifically modified at the DSB site and NuA4 is efficiently recruited at the DSB site to acetylate histone H4. By contrast, the eaf3-W84A/W88A yng2-PHD double mutant dramatically reduced the enrichment of H4K12ac at the DSB, suggesting the impairment of NuA4 recruitment. H2A-S129p activation is prolonged at DSB, suggesting the double mutant impairs DSB repair. Our results suggest that the DSB can induce the specific H2AS129p, H3K4me2, H3K4me3, and H3K36me2 modifications at DSB, which in turns recruits NuA4 complex by the multiple interactions between these modified histones and subunits of NuA4. These interactions include H2AS129p-Arp4, H3K36me2-chromo domain and H3K4me3-PHD interactions and these multiple interactions strengthen the recruitment of NuA4 at DSBs.

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