Johnson K A, Mierzwa M L, Fink S P, Marnett L J
A. B. Hancock, Jr. Memorial Laboratory for Cancer Research, Department of Biochemistry and Chemistry, Center in Molecular Toxicology, Vanderbilt Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
J Biol Chem. 1999 Sep 17;274(38):27112-8. doi: 10.1074/jbc.274.38.27112.
The ability of the methyl-directed mismatch repair system to recognize and repair the exocyclic adducts propanodeoxyguanosine (PdG) and pyrimido[1,2-alpha]purin-10(3H)-one (M(1)G), the major adduct derived from the endogenous mutagen malondialdehyde, has been assessed both in vivo and in vitro. Both adducts were site-specifically incorporated into M13MB102 DNA, and the adducted genomes were electroporated into wild-type or mutS-deficient Escherichia coli strains. A decrease in mutations caused by both adducts was observed in mutS-deficient strains, suggesting that MutS was binding to the adducts and blocking repair by nucleotide excision repair. This hypothesis was supported by the differences in mutation frequency observed when hemimethylated genomes containing PdG on the (-)-strand were electroporated into a uvrA(-) strain. The ability of purified MutS to bind to PdG- or M(1)G-containing 31-mer duplexes in vitro was assessed using both surface plasmon resonance and gel shift assays. MutS bound to M(1)G:T-containing duplexes with similar affinity to a G:T mismatch but less strongly to M(1)G:C- and PdG-containing duplexes. Dissociation from each of the adduct-containing duplexes occurred at a faster rate than from a G:T mismatch. The present results indicate that MutS can bind to exocyclic adducts resulting from endogenous DNA damage and trigger their removal by mismatch repair or protect them from removal by nucleotide excision repair.
甲基定向错配修复系统识别和修复环外加合物丙烷脱氧鸟苷（PdG）和嘧啶并[1,2-α]嘌呤-10（3H）-酮（M(1)G）的能力已在体内和体外进行了评估，M(1)G是内源性诱变剂丙二醛产生的主要加合物。两种加合物都被位点特异性地掺入到M13MB102 DNA中，并且将含有加合物的基因组电穿孔导入野生型或mutS缺陷型大肠杆菌菌株。在mutS缺陷型菌株中观察到由两种加合物引起的突变减少，这表明MutS与加合物结合并通过核苷酸切除修复阻断修复。当将在（-）链上含有PdG的半甲基化基因组电穿孔导入uvrA（-）菌株时观察到的突变频率差异支持了这一假设。使用表面等离子体共振和凝胶迁移分析评估了纯化的MutS在体外与含有PdG或M(1)G的31聚体双链体结合的能力。MutS与含有M(1)G:T的双链体结合的亲和力与G:T错配相似，但与含有M(1)G:C和PdG的双链体结合较弱。从每种含有加合物的双链体上解离的速度比从G:T错配上解离的速度快。目前的结果表明，MutS可以与内源性DNA损伤产生的环外加合物结合，并通过错配修复触发其去除，或保护它们不被核苷酸切除修复去除。
