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DNA mismatch repair (MMR) system corrects mismatched bases that are generated mainly by DNA replication errors. In the early stages of MMR, MutL endonuclease incises the errorcontaining strand of the duplex to initiate the downstream excision reaction that is followed by resynthesis of the strand. In this study, purified MutL from Rhodobacter sphaeroides (rsMutL) was shown possess an endonuclease activity. Based on the alignment of 1483 homologs of MutL from bacteria, we evaluated the conserved functional motifs present. The sequence of rsMutL C-domain was shown to contain 5 motifs comprising rsMutL catalytic center responsible for DNA cleavage including metal-binding sites, while 7 conservative motifs related to ATP binding and hydrolysis specific to GHKL-family of ATPases were present in rsMutL N-domain. The analysis of sequence and biochemical properties for 5 studied homologues of MutL with endonuclease function revealed that MutL from Neisseria gonorrhoeae (ngMutL) is most similar to rsMutL. The presence of ATP inhibits DNA cleavage induced by MutL from both organisms. Mg2+ promotes the nicking of plasmid DNA by rsMutL with the highest efficiency. The presence of Mg2+ and Mn2+ in the reaction mixture leads to a significant increase in quantity of linear plasmid form, probably due to the activation of the catalytic center of the second MutL subunit. Zn2+ attributed to structural function in MutL homologues was shown to suppress rsMutL and ngMutL endonuclease activity in the presence of excess Mn2+. The ability of rsMutL and ngMutL to interact with DNA and ATP has been investigated. Complex formation of rsMutL with duplexes of varying length from 4 to 76 base pairs was not observed, while ngMutL formed a complex with a 30-base pair duplex. The KM values of the ATP hydrolysis reaction by the ngMutL protein are almost two times lower than for rsMutL, indicating a higher affinity of ngMutL to ATP. However, rsMutL hydrolyzed ATP an order of magnitude faster than ngMutL. Perhaps, this causes a large conformational mobility of rsMutL, which does not allow stable formation of a short-lived DNA-protein complex. The affinity modification of the ngMutL by reactive DNA containing the pyridyldisulfide group demonstrated the proximity of Cys residues of the catalytic center of the enzyme to the DNA ligand. The work was supported by grant No 17-54-45126 IND_a from the Russian Foundation for Basic Research.