Double strand-breaks (DSBs) of genomic DNA caused by ionizing radiation or mutagenic chemicals are a common source of mutation, recombination, chromosomal aberration, and cell death. Linker histones are DNA packaging proteins with established roles in chromatin compaction, gene transcription, and in homologous recombination (HR)-mediated DNA repair. Using a machine-learning model for functional prioritization of eukaryotic post-translational modifications (PTMs) in combination with genetic and biochemical experiments with the yeast linker histone, Hho1, we discovered that site-specific phosphorylation sites regulate HR and HR-mediated DSB repair. Five total sites were investigated (T10, S65, S141, S173, and S174), ranging from high to low function potential as determined by the model. Of these, we confirmed S173/174 are phosphorylated in yeast by mass spectrometry and found no evidence of phosphorylation at the other sites. Phospho-nullifying mutations at these two sites results in a significant decrease in HR-mediated DSB repair templated either with oligonucleotides or a homologous chromosome, while phospho-mimicing mutations have no effect. S65, corresponding to a mammalian phosphosite that is conserved in yeast, exhibited similar effects. None of the mutations affected base- or nucleotide-excision repair, nor did they disrupt non-homologous end joining or RNA-mediated repair of DSBs when sequence heterology between the break and repair template strands was low. More extensive analysis of the S174 phospho-null mutant revealed that its repression of HR and DSB repair is proportional to the degree of sequence heterology between DSB ends and the HR repair template. Taken together, these data demonstrate the utility of machine learning for the discovery of functional PTM hotspots, reveal linker histone phosphorylation sites necessary for HR and HR-mediated DSB repair, and provide insight into the context-dependent control of DNA integrity by the yeast linker histone Hho1.

中文翻译：

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接头组蛋白PTM热点的系统分析显示，磷酸化位点可调节同源重组和DSB修复。

电离辐射或诱变化学物质引起的基因组DNA双链断裂（DSB）是突变，重组，染色体畸变和细胞死亡的常见来源。接头组蛋白是DNA包装蛋白，在染色质紧实，基因转录和同源重组（HR）介导的DNA修复中具有确定的作用。使用机器学习模型对真核翻译后修饰（PTMs）进行功能优先排序，并结合酵母连接蛋白组蛋白Hho1的遗传和生化实验，我们发现位点特异性磷酸化位点可调节HR和HR介导的DSB修复。根据模型确定，调查了五个总位点（T10，S65，S141，S173和S174），范围从高到低。这些，我们通过质谱法证实S173 / 174在酵母中被磷酸化，而在其他位点没有发现磷酸化的迹象。在这两个位点的磷酸化突变导致以寡核苷酸或同源染色体为模板的HR介导的DSB修复显着降低，而模拟磷酸化的突变则没有效果。对应于酵母中保守的哺乳动物磷酸酯的S65表现出相似的作用。当断裂和修复模板链之间的序列异质性较低时，这些突变均不会影响碱基或核苷酸切除修复，也不会破坏非同源末端连接或RNA介导的DSB修复。对S174磷酸空突变体的更广泛分析显示，其对HR和DSB修复的抑制与DSB末端与HR修复模板之间的序列异源度成正比。综上所述，这些数据证明了机器学习在发现功能性PTM热点方面的实用性，揭示了HR和HR介导的DSB修复所必需的接头组蛋白磷酸化位点，并提供了由酵母接头对上下文依赖的DNA完整性控制的见解。组蛋白Hho1。