Sop1和Sop2的功能及其在DNA损伤应答中的作用

DNA damage response (DDR) plays role in maintaining genome integrity. It's processes must be understood in chromatin context. Eukaryotic genomic DNA is folded with histones and non-histone proteins into chromatin. The covalent modifications of histone play an important role in DNA damage response, since they affect the compaction of chromatin and recruitment of DNA damage response or repair proteins. It has been shown that H4K20me2/H4K20me1 is involved in DNA damage checkpoint activation by recruiting Crb2 which is a checkpoint protein. We found that PWWP domain protein Pdp1 associates with H4K20 methyltransferase Set9 and regulates H4K20 methylation in fission yeast Schizosaccharomyces pombe. Pdp1 deletion mutant is sensitive to DNA damage reagents. To further investigate the mechanism underlying the regulation of DNA damage response, we screened fission yeast deletion library for the suppressors that can rescue the DNA damage sensitive phenotype of Pdp1 deletion mutant. After genome-wide screening, we have got at least 12 hits. We will focus on the molecular mechanisms of two hits, Sop1 and Sop2, by complex purification followed by mass spec and checking histone modifications, phosphorylation of checkpoint proteins, cell cycle changes upon DNA damage treatment, DNA recombination and chromatin compaction of the deletion mutants. We found that sop1Δ and sop2Δ are also sensitive to DNA damage treatment. Based on the domain predication, these two unknown genes might play a role in nucleosome assembly and protein transport respectively. Dissecting the functions of these protein complexes will help us get better understanding of the functions of Sop1 and Sop2 in the DNA damge response and maintenance of genome stability.

DNA损伤应答是一个复杂的信号传递过程，一些修饰的组蛋白也参与其中。裂殖酵母中H4K20甲基化通过招募Crb2蛋白，参与DNA损伤检验点的激活。Pdp1蛋白调节H4K20甲基化。Pdp1基因缺失突变体检测不到H4K20甲基化，对紫外线等DNA损伤处理敏感。本研究利用裂殖酵母缺失突变体库，筛选抗紫外线的Pdp1突变抑制因子。目前已筛到至少12个突变体，并克隆了其中两个基因Sop1和Sop2，它们编码的蛋白定位在细胞核，功能未知。初步研究发现，这两个基因的突变体本身对DNA损伤敏感。功能域预测显示，Sop1和Sop2可能分别参与核小体组装和组蛋白及其它蛋白的核内外转运。我们将主要针对这两个基因，通过纯化蛋白复合物、复合物功能分析以及遗传和生化方法，研究它们对染色质结构、DNA损伤应答和修复等方面的影响。这些结果将有助于从染色质水平解析裂殖酵母中DNA损伤和修复的调控网络。