RPA ( replication protein A ) 為一普遍存於真核細胞中之單股DNA結合蛋白，主要由RPA1、RPA2、RPA3三個subunits組成一穩定複合物。其功能主要和DNA之複製、修補、以及重組有關。RPA除了可與單股DNA結合外，亦和其他蛋白間有交互作用。過去研究顯示，RPA可與DNA polymerase α 及其它複製相關蛋白質結合，或是與XPA、XPG及uracil-DNA glycosylase 等與DNA修補相關之蛋白結合。此外，RPA2在細胞週期S phase時期有磷酸化現象。而當DNA損傷時，RPA2會因細胞內一些kinase之作用而有高度磷酸化現象。然而其真正的作用機制目前尚不清楚。因此，在先前的研究中，我們利用yeast-two-hybrid 之方法，篩選可能和磷酸化RPA2蛋白有交互作用之因子。初步選殖出有交互作用者為核糖蛋白L5。我們發現在in vitro 或in vivo下L5皆會與高度磷酸化之RPA2結合，而且complex裏還包含了RPA1。我們也證明了另一個 L5的結合蛋白MDM2在紫外光照射細胞後與L5的結合量會提升，而且MDM2必須在L5及磷酸化的RPA存在下才能與L5-磷酸化RPA產生交互作用；當我們以RNA干擾技術抑制L5的表現時，MDM2則不存在於磷酸化RPA complex裏。有趣的是我們發現L5 knock down會減弱p53受UV照射後serine 46位置的磷酸化，而且這個現象可在ARF不表現的情況下發生，但是serine 15位置的磷酸化則不受影響。最後我們發現L5在UV刺激後在細胞的位置會由核仁轉移到核質。這些結果證明了RPA2的高度磷酸化可能吸引了L5-MDM2 complex進而活化p53的damage response pathway，而L5在其中扮演了重要的角色。

　Replication protein a (RPA) is a three subunits single-stranded DNA binding protein required for DNA replication, repair and recombination. RPA interacts specifically with essential repair proteins (e.g. XPA, XPG, and uracil-DNA glycosylase) and is required for loading of DNA polymerase α and additional replication fork proteins. In addition, the middle subunit of RPA (RPA2) is phosphorylated during the cell cycle S-phase and becomes hyper-phosphorylated in response to DNA damage. But its significance in the signaling of damage response pathway is poorly understood. Our previous studies found that a ribosomal protein L5 could interact with a truncated form of RPA2 in yeast two-hybrid system. Here we showed that the L5 protein specifically binds to hyperphosphorylated RPA2 in vitro and in vivo and this complex also contains RPA1. We demonstrated that the protein levels of L5 binding to the MDM2, a negative regulator of tumor suppressor p53, were decreased at the first 0.5 hr but subsequently were increased after UV irradiation. Moreover, MDM2 also associates with the L5-phosphorylated RPA protein complex in the L5 and phosphorylated RPA-dependent manner. Interestingly, the phosphorylation of p53 at serine 46, but not serine 15, was prevented by L5 small interfering RNA, and this inhibition is ARF independent. Finally, a fractionation assay revealed that the localization of L5 is changed from nucleolous to nucleoplasm upon UV treatment. Taken together, these results suggest that hyperphosphorylated RPA2 recruits L5-MDM2 complex to lead to the activation of p53 in response to UV damage.

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