Funakoshi Masafumi, Nambara Daisuke, Hayashi Yuichiro, Zhang-Akiyama Qiu-Mei
Laboratory of Stress Response Biology, Division of Biological Sciences, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto, 606-8502 Japan.
Genes Environ. 2017 Dec 1;39:27. doi: 10.1186/s41021-017-0087-7. eCollection 2017.
Apurinic/apyrimidinic (AP) sites are one of the most frequent DNA lesions. AP sites inhibit transcription and DNA replication, and induce cell death. AP endonucleases are key enzymes in AP site repair. Several types of AP endonucleases have been reported, such as AP endonuclease 2 (APEX2) and ribosomal protein P0 (P0). However, it is not known how the functions and roles differ among AP endonucleases. To clarify the difference of roles among AP endonucleases, we conducted biochemical analysis focused on APEX2 and P0 homologues in . Amino acid sequence analysis suggested that CiAPEX2 and CiP0 are AP endonuclease homologues. Although we could not detect AP endonuclease or 3'-phosphodiesterase activity, these two purified proteins exhibited 3'-5' exonuclease activity. This 3'-5' exonuclease activity was sensitive to ethylenediaminetetraacetic acid (EDTA), and the efficiency of this activity was influenced by the 3'-terminus of substrate DNA. Both CiAPEX2 and CiP0 degraded not only a 5'-protruding DNA end, but also nicked DNA, which is generated through AP endonuclease 1 (APEX1) cleavage. These two genes partially complemented the growth rate of AP endonuclease-deficient treated with hydrogen peroxide. These results indicate that 3'-5' exonuclease activity is an evolutionarily conserved enzymatic activity of APEX2 and P0 homologues and this enzymatic activity may be important for AP endonucleases.
脱嘌呤/脱嘧啶（AP）位点是最常见的DNA损伤之一。AP位点会抑制转录和DNA复制，并诱导细胞死亡。AP内切核酸酶是AP位点修复中的关键酶。已经报道了几种类型的AP内切核酸酶，如AP内切核酸酶2（APEX2）和核糖体蛋白P0（P0）。然而，尚不清楚AP内切核酸酶之间的功能和作用有何不同。为了阐明AP内切核酸酶之间作用的差异，我们针对APEX2和P0同源物进行了生化分析。氨基酸序列分析表明，CiAPEX2和CiP0是AP内切核酸酶同源物。尽管我们未检测到AP内切核酸酶或3'-磷酸二酯酶活性，但这两种纯化的蛋白均表现出3'-5'外切核酸酶活性。这种3'-5'外切核酸酶活性对乙二胺四乙酸（EDTA）敏感，并且该活性的效率受底物DNA 3'-末端的影响。CiAPEX2和CiP0不仅能降解5'-突出的DNA末端，还能降解通过AP内切核酸酶1（APEX1）切割产生的带切口的DNA。这两个基因部分弥补了用过氧化氢处理的AP内切核酸酶缺陷型的生长速率。这些结果表明，3'-5'外切核酸酶活性是APEX2和P0同源物在进化上保守的酶活性，并且这种酶活性可能对AP内切核酸酶很重要。
