Wang Pingping, Byrum Stephanie, Fowler Faith C, Pal Sangita, Tackett Alan J, Tyler Jessica K
Weill Cornell Medicine, Department of Pathology and Laboratory Medicine, New York, NY 10065, USA.
Genes and Development Graduate Program of the University of Texas MD Anderson Cancer Center, UT Health Graduate School of Biomedical Sciences, Houston, TX 77030, USA.
Nucleic Acids Res. 2017 Nov 2;45(19):10923-10940. doi: 10.1093/nar/gkx844.
Here, we use ChAP-MS (chromatin affinity purification with mass spectrometry), for the affinity purification of a sequence-specific single-copy endogenous chromosomal locus containing a DNA double-strand break (DSB). We found multiple new histone post-translational modifications enriched on chromatin bearing a DSB from budding yeast. One of these, methylation of histone H3 on lysine 125, has not previously been reported. Among over 100 novel proteins enriched at a DSB were the phosphatase Sit4, the RNA pol II degradation factor Def1, the mRNA export protein Yra1 and the HECT E3 ligase Tom1. Each of these proteins was required for resistance to radiomimetics, and many were required for resistance to heat, which we show here to cause a defect in DSB repair in yeast. Yra1 and Def1 were required for DSB repair per se, while Sit4 was required for rapid inactivation of the DNA damage checkpoint after DSB repair. Thus, our unbiased proteomics approach has led to the unexpected discovery of novel roles for these and other proteins in the DNA damage response.
在这里，我们使用染色质亲和纯化质谱法（ChAP-MS）来亲和纯化含有DNA双链断裂（DSB）的序列特异性单拷贝内源性染色体位点。我们发现了多种新的组蛋白翻译后修饰，这些修饰在来自芽殖酵母的带有DSB的染色质上高度富集。其中之一，即组蛋白H3赖氨酸125位点的甲基化，此前尚未见报道。在超过100种在DSB处高度富集的新蛋白中，有磷酸酶Sit4、RNA聚合酶II降解因子Def1、mRNA输出蛋白Yra1和HECT E3连接酶Tom1。这些蛋白中的每一种都是抗辐射模拟剂所必需的，而且许多蛋白也是耐热所必需的，我们在此表明热会导致酵母中DSB修复出现缺陷。Yra1和Def1本身就是DSB修复所必需的，而Sit4是DSB修复后DNA损伤检查点快速失活所必需的。因此，我们的无偏差蛋白质组学方法意外地发现了这些蛋白及其他蛋白在DNA损伤反应中的新作用。
