利用串联亲和纯化的方法筛选耻垢分枝杆菌中MutM的相互作用蛋白

Mut M(formamidopyrimidine-DNA glycosylase,Fpg)是原核生物碱基切除修复系统(BER)中同时具有DNA糖苷酶和脱嘌呤/脱嘧啶AP裂解酶活性的一种双功能酶,不但可以识别DNA损伤,而且能切除损伤的碱基,从而参与到许多种损伤的修复过程.除了高致突变率的8-羟基鸟嘌呤(8-oxoguanine,8-oxo G)外,Mut M在其他损伤修复中具体作用机制还不清楚.本研究主要以耻垢分枝杆菌(M.smegmatis)为研究对象,利用串联亲和纯化技术和质谱相结合的方法对可能与Mut M相互作用的蛋白因子进行发现和鉴定,并于体外用Far-western和GST pull-down方法对鉴定出的蛋白DEAD-box rna helicase、Rps C、Uvr A与Mut M的相互作用进行了验证.实验结果表明,利用串联亲和纯化方法来发现Mut M相互作用的蛋白是切实可行的.本研究为进一步深入研究Mut M在其参与的损伤修复中的具体机制提供了切入点.

MutM Interaction Partners Detected in Mycobacterium smegmatis by Tandem Affinity Purification

MutM (Formamidopyrimidine-DNA glycosylase, Fpg), a bifunctional base excision repair enzyme (DNA glycosylase/AP lyase), is involved in the repair of many kinds of DNA damage, including the formation of 8-oxoguanine, 5-formyluracil, and C/C mismatches, through recognizing DNA damage and removing damaged bases. The mechanisms of MutM involvement, however, with the exception of 8-oxoG, are poorly understood. Here, we identified proteins which interact with MutM in Mycobacterium smegmatis using tandem affinity purification and mass spectrometry and used Far-western and GST pull-down analysis to validate the interactions between MutM and DEAD-box rna helicase, RpsC, and UvrA. Results demonstrated that tandem affinity purification is a suitable method for identifying MutM interacting proteins and provided insights into the mechanism by which MutM is involved in DNA damage repair.