Proteomics analysis of hEPC1 polycomb protein complex in DNA damaged repair.

真核生物的基因组是紧密堆积在染色质中的，染色质是一个由DNA、组蛋白和非组蛋白组成的有层次的复合体。这种包装是大多数DNA功能的常见障碍。最近，我们发现组蛋白乙酰化酶复合物TIP 60参与DNA修复和细胞凋亡。然而，TIP 60复合物如何参与DNA修复仍不清楚。为了更好地理解TIP 60复合物在DNA修复中的作用机制，从DNA损伤后的染色质可溶性组分中纯化TIP 60复合物。结果，TIP 60复合物的组分polycomb增强子（hEPC 1）与TIP 60复合物解离。为了阐明hEPC 1在DNA修复中的作用，我们从HeLa细胞中纯化了hEPC 1。hEPC 1被包含在多蛋白复合物中。MS/MS分析表明，hEPC 1复合物中含有激酶、甲基转移酶、组蛋白H2 AX、TIP 60组蛋白乙酰化酶和泛素相关蛋白。观察到甲基转移酶活性。此外，组蛋白H2 AX在DNA损伤后被磷酸化。在表达siEPC RNA的HeLa细胞中，H2 AX的磷酸化被抑制，表明hEPC 1复合物包含激酶结构域。

Eukaryotic genome is the tightly packed into the chromatin, a hierarchically organized complex of DNA and histone and nonhistone proteins. This packing represents a common obstacle for most of the DNA functions. Recently, we revealed that TIP60 histone acetylase complex involved in DNA repair and apoptosis. However, it remains unknown how TIP60 complex involves in DNA repair. To better understand the mechanism of TIP60 complex in DNA repair, TIP60 complex purifies from chromatin soluble fraction after DNA damage. As a result, enhancer of polycomb (hEPC1), the component of TIP60 complex, dissociated with the TIP60 complex. In order to clarify the role of hEPC1 in DNA repair,We purified the hEPC1 from HeLa after induction of DNA damage. hEPC1 was included in multiple protein complex. MS/MS analysis indicated that kinase, metyltransferase enzyme, histone H2AX, TIP60 histone acetylase and ubiquitin related proteins were identified as a component of hEPC1 complex. Metyltransferase activity was observed. In addition, Histone H2AX is phosphorylated after DNA damage. The phosphorylation of H2AX was inhibited in HeLa cells expressing siEPC RNA suggested that the hEPC1 complex includes kinase domain.