Live cell analysis of cellular responses to various types of DNA damage induced by reactive oxygen

利用UVA激光通过显微透镜局部照射，我们开发了在人类细胞核内产生各种类型的氧化DNA损伤的方法，并分析了蛋白质对损伤部位的实时反应。我们确定了一些新的蛋白质参与修复碱基损伤，单链断裂和双链断裂。此外，我们采用LC/MS/MS谱仪进行蛋白质组学分析，鉴定了人体细胞中氧化DNA损伤修复所需的蛋白质复合物。DNA损伤导致基因组不稳定和细胞死亡，但许多细胞对DNA损伤的反应仍然是难以捉摸的。例如，我们发现。一种人类蛋白，PALF （PNK和apx样FHA蛋白），具有FHA（叉头相关）结构域和新型锌指状CYR（半胱氨酸-酪氨酸-精氨酸）基元，参与对DNA损伤的反应。我们发现CYR基序广泛分布于高等真核生物的DNA修复蛋白中，并且PALF以及具有串联CYR基序的果蝇蛋白具有针对碱基位点和其他类型碱基损伤的内切酶和外切酶活性。在人类细胞中，PALF以一种依赖于聚（adp -核糖）聚合酶1 （PARP1）的方式在单链断裂处迅速积累。事实上，PALF直接与PARP1相互作用，并且是其激活和细胞对甲基甲烷磺酸盐的抗性所必需的。PALF还直接与KU86、LIGASEIV和磷酸化的XRCC4蛋白相互作用，并在突出的DNA末端具有内切酶/外切酶活性。产生双链断裂的各种处理诱导PALF灶的形成，PALF灶与gammaH2AX灶完全重合。因此，PALF和CYR基序可能在高等真核生物的DNA修复中发挥重要作用。

Using local irradiation of UVA laser light through microscopic lens, we have developed methods to produce various types of oxidative DNA damage within human nucleus and analyzed real time response of proteins to the damage site. We identified a number of novel proteins involved in the repair of base damage, single-strand breaks and double-strand breaks. Furthermore, we applied proteome analysis by LC/MS/MS spectrometer and identified protein complexes necessary for the repair of oxidative DNA damage within human cell. DNA damage causes genome instability and cell death, but many of the cellular responses to DNA damage still remain elusive. We found, for example. a human protein, PALF (PNK and APTX-like FHA protein), with an FHA (forkhead-associated) domain and novel zinc-finger-like CYR (cysteine-tyrosine-arginine) motifs that are involved in responses to DNA damage. We found that the CYR motif is widely distributed among DNA repair proteins of higher eukaryotes, and that PALF, as well as a Drosophila protein with tandem CYR motifs, has endo- and exonuclease activities against abasic site and other types of base damage. PALF accumulates rapidly at single-strand breaks in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner in human cells. Indeed, PALF interacts directly with PARP1 and is required for its activation and for cellular resistance to methyl-methane sulfonate. PALF also interacts directly with KU86, LIGASEIV and phosphorylated XRCC4 proteins and possesses endo/exonuclease activity at protruding DNA ends. Various treatments that produce double-strand breaks induce formation of PALF foci, which fully coincide with gammaH2AX foci. Thus, PALF and the CYR motif may play important roles in DNA repair of higher eukaryotes.