School of Biological Sciences, Georgia Institute of Technology, 950 Atlantic Drive NW Atlanta GA 30332,USA.
School of Biological Sciences, Georgia Institute of Technology, 950 Atlantic Drive NW Atlanta GA 30332,USA; School of Chemistry and Biochemistry, Georgia Institute of Technology, 950 Atlantic Drive NW Atlanta GA 30332,USA.
DNA Repair (Amst). 2020 Feb;86:102763. doi: 10.1016/j.dnarep.2019.102763. Epub 2019 Nov 29.
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.
双链断裂（DSBs）是由电离辐射或诱变化学物质引起的基因组 DNA 断裂，是突变、重组、染色体畸变和细胞死亡的常见来源。连接组蛋白是 DNA 包装蛋白，在染色质紧缩、基因转录以及同源重组（HR）介导的 DNA 修复中具有既定作用。我们使用用于真核后翻译修饰（PTM）功能优先级排序的机器学习模型，结合酵母连接组蛋白 Hho1 的遗传和生化实验，发现特定的磷酸化位点调节 HR 和 HR 介导的 DSB 修复。共研究了五个总位点（T10、S65、S141、S173 和 S174），根据模型确定了从高到低的功能潜力。其中，我们通过质谱法证实酵母中 S173/174 发生磷酸化，并且没有在其他位点发现磷酸化的证据。这两个位点的磷酸化缺失突变会导致 HR 介导的 DSB 修复模板的显著减少，无论是使用寡核苷酸还是同源染色体，而磷酸化模拟突变则没有影响。S65 对应于酵母中保守的哺乳动物磷酸化位点，表现出类似的效果。当断裂和修复模板之间的序列异质性较低时，这些突变均不影响碱基或核苷酸切除修复，也不影响非同源末端连接或 DSB 的 RNA 介导修复。对 S174 磷酸化缺失突变体的更广泛分析表明，其对 HR 和 DSB 修复的抑制作用与 DSB 末端和 HR 修复模板之间的序列异质性程度成正比。总之，这些数据表明机器学习在发现功能 PTM 热点方面的实用性，揭示了连接组蛋白磷酸化位点对 HR 和 HR 介导的 DSB 修复的必要性，并深入了解酵母连接组蛋白 Hho1 对 DNA 完整性的上下文依赖性控制。
