Arabidopsis ARP endonuclease functions in a branched base excision DNA repair pathway completed by LIG1
Arabidopsis ARP endonuclease functions in a branched base excision DNA repair pathway completed by LIG1
复制标题
DOI：
10.1111/j.1365-313x.2011.04720.x
复制
发表时间：
2011-11-01
期刊：
影响因子：
7.2
通讯作者：
Ariza, Rafael R.
中科院分区：
文献类型：
--
作者：
Cordoba-Canero, Dolores;Roldan-Arjona, Teresa;Ariza, Rafael R.
碱基切除修复（BER）是植物细胞防御DNA损伤的一种重要机制，但目前对它的研究还不多。我们使用了一种检测方法，监测修复受损的基地和无碱基（脱嘌呤/脱嘧啶，AP）的网站在拟南芥的特点后切除事件在植物BER。我们发现，在拟南芥细胞提取物中，脱嘌呤核酸内切酶-氧化还原蛋白（阿普）是主要的AP核酸内切酶活性，并且是体外尿嘧啶BER过程中AP切割所必需的。缺乏阿普的突变植物生长正常，但对5-氟尿嘧啶（一种有利于尿嘧啶错误掺入DNA的化合物）过敏。我们还发现，在AP切口后，单核苷酸或长补丁DNA合成（SN-或LP-BER）之间的选择受到修复缺口的5'端的影响。当5'端被阻断并且不适于β-消除时，SN子途径被废除，并且仅通过LP-BER完成修复。最后，我们提供的证据表明，拟南芥DNA连接酶I（LIG 1）是必需的SN-和LP-BER。lig 1 RNAi沉默的细胞系显示尿嘧啶BER非常降低，并且抗LIG 1抗体消除了野生型细胞提取物中的修复。相反，敲除lig 4（-/-）突变体表现出正常的BER和切口连接水平。我们的研究结果表明，分支BER途径完成的DNA连接酶I家族的成员可能是一个古老的功能，在真核生物。
Base excision repair (BER) is an essential cellular defence mechanism against DNA damage, but it is poorly understood in plants. We used an assay that monitors repair of damaged bases and abasic (apurinic/apyrimidinic, AP) sites in Arabidopsis to characterize post-excision events during plant BER. We found that Apurinic endonuclease-redox protein (ARP) is the major AP endonuclease activity in Arabidopsis cell extracts, and is required for AP incision during uracil BER in vitro. Mutant plants that are deficient in ARP grow normally but are hypersensitive to 5-fluorouracil, a compound that favours mis-incorporation of uracil into DNA. We also found that, after AP incision, the choice between single-nucleotide or long-patch DNA synthesis (SN- or LP-BER) is influenced by the 5' end of the repair gap. When the 5' end is blocked and not amenable to beta-elimination, the SN sub-pathway is abrogated, and repair is accomplished through LP-BER only. Finally, we provide evidence that Arabidopsis DNA ligase I (LIG1) is required for both SN- and LP-BER. lig1 RNAi-silenced lines show very reduced uracil BER, and anti-LIG1 antibody abolishes repair in wild-type cell extracts. In contrast, knockout lig4(-/-) mutants exhibit normal BER and nick ligation levels. Our results suggest that a branched BER pathway completed by a member of the DNA ligase I family may be an ancient feature in eukaryotic species.