Characterization of new identified novel human AP endonuclease

DUF2228是一个功能未知的保守蛋白家族，广泛分布于包括人类在内的各种脊椎动物中。在昆虫和蠕虫的一部分中，DUF2228蛋白在N-末端含有一个额外的结构域，包含一个CyR基序(C2H2锌指基序)。由于CyR基序首先在人类修复蛋白PALF(ALPF)中被发现，并在DNA代谢的各种蛋白中发现，如DNA修复和DNA损伤检查点，在其N端或C端，我们推测DUF2228家族蛋白也具有与DNA代谢相关的功能。我们分离了果蝇CG1218-PA和人C4orf27(APNX)属于DUF2228的基因，并对其进行了鉴定。在这里，我们发现：1)果蝇CG1218-PA蛋白以多聚ADP核糖依赖的方式聚集在DNA损伤部位。2)缺乏CyR基序的人APNX蛋白与PARP1形成异源二聚体，并以PARP1依赖的方式聚集在DNA损伤部位。3)CG1218-PA和APNX对碱性位点具有核酸内切酶和核酸外切酶活性。4)在HeLa细胞中使用siRNA抑制APNX的表达使细胞对甲烷磺酸甲酯敏感，甲基甲烷磺酸产生甲基化碱基导致单链断裂。这些数据表明，DUF2228蛋白CG1218-PA和APNX在DNA碱基损伤修复和单链断裂中发挥重要作用。

DUF2228 is a functionally unknown, conserved protein family distributed from worm to vertebrate including human. In insect and a part of worm DUF2228 protein has an additional domain containing a CYR motif (C2H2 zinc finger motif)in the N-terminal region. Since CYR motif was first identified in the human repair protein, PALF (ALPF), and found in various proteins of DNA metabolism, such as DNA repair and the DNA damage checkpoint, at their N-or C-termini, we hypothesized that DUF2228 family proteins have also DNA metabolism related functions. We isolated cDNA of Drosophila CG1218-PA and human C4orf27(APNX)belonging to DUF2228 and characterized these proteins. Here we show that ; 1)a Drosophila CG1218-PA protein accumulates at DNA damaged site in a poly-ADP-ribose-dependent manner. 2)Human APNX protein that lacks a CYR motif forms a hetero dimmer with PARP1 and accumulates at DNA damaged site in a PARP1-dependent manner. 3)CG1218-PA and APNX possess endo-and exonuclease activities against abasic site. 4)Suppression of the expression of APNX using siRNA in HeLa cells provided the cells with sensitivity to methyl methane sulfonate, which produces methylated bases leading to single-strand breaks. These data suggest that DUF2228 protein CG1218-PA and APNX play important roles in the repair of DNA base damage and single-strand breaks.