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真实的时间响应。我们发现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，我们应用LC/MS/MS光谱仪进行蛋白质组分析，并确定了人类细胞内氧化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损伤的反应仍然难以捉摸。我们发现，例如。一种人类蛋白质PALF（PNK和APTX样FHA蛋白），具有FHA（叉头相关）结构域和新型锌指样CYR（半胱氨酸-酪氨酸-精氨酸）基序，参与对DNA损伤的应答。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脱碱基位点和其他类型的碱基损伤的内切核酸酶和外切核酸酶活性。PALF在人细胞中以聚（ADP-核糖）聚合酶1（PARP 1）依赖性方式在单链断裂处快速积累。事实上，PALF直接与PARP 1相互作用，并且是其活化和细胞对甲基甲烷磺酸盐的抗性所必需的。PALF还与KU 86、LIGASEIV和磷酸化XRCC 4蛋白直接相互作用，并在突出的DNA末端具有内切/外切核酸酶活性。产生双链断裂的各种处理诱导PALF病灶的形成，其与γ H2 AX病灶完全一致。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.