Monakhova M V, Penkina A I, Pavlova A V, Lyaschuk A M, Kucherenko V V, Alexeevski A V, Lunin V G, Friedhoff P, Klug G, Oretskaya T S, Kubareva E A
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119991, Russia.
Biochemistry (Mosc). 2018 Mar;83(3):281-293. doi: 10.1134/S0006297918030082.
We have purified the MutL protein from Rhodobacter sphaeroides mismatch repair system (rsMutL) for the first time. rsMutL demonstrated endonuclease activity in vitro, as predicted by bioinformatics analysis. Based on the alignment of 1483 sequences of bacterial MutL homologs with presumed endonuclease activity, conserved functional motifs and amino acid residues in the rsMutL sequence were identified: five motifs comprising the catalytic site responsible for DNA cleavage were found in the C-terminal domain; seven conserved motifs involved in ATP binding and hydrolysis and specific to the GHKL family of ATPases were found in the N-terminal domain. rsMutL demonstrated the highest activity in the presence of Mn2+. The extent of plasmid DNA hydrolysis declined in the row Mn2+ > Co2+ > Mg2+ > Cd2+; Ni and Ca2+ did not activate rsMutL. Divalent zinc ions inhibited rsMutL endonuclease activity in the presence of Mn2+ excess. ATP also suppressed plasmid DNA hydrolysis by rsMutL. Analysis of amino acid sequences and biochemical properties of five studied bacterial MutL homologs with endonuclease activity revealed that rsMutL resembles the MutL proteins from Neisseria gonorrhoeae and Pseudomonas aeruginosa.
我们首次从球形红杆菌错配修复系统（rsMutL）中纯化出了MutL蛋白。如生物信息学分析所预测的那样，rsMutL在体外表现出核酸内切酶活性。基于1483个具有假定核酸内切酶活性的细菌MutL同源物序列的比对，在rsMutL序列中鉴定出了保守的功能基序和氨基酸残基：在C末端结构域中发现了五个构成负责DNA切割的催化位点的基序；在N末端结构域中发现了七个参与ATP结合和水解且特定于ATP酶GHKL家族的保守基序。rsMutL在Mn2+存在时表现出最高活性。质粒DNA水解程度按Mn2+ > Co2+ > Mg2+ > Cd2+的顺序下降；Ni和Ca2+不能激活rsMutL。在Mn2+过量存在时，二价锌离子抑制rsMutL核酸内切酶活性。ATP也抑制rsMutL对质粒DNA的水解。对五个具有核酸内切酶活性的已研究细菌MutL同源物的氨基酸序列和生化特性分析表明，rsMutL类似于淋病奈瑟菌和铜绿假单胞菌的MutL蛋白。
