[MutL Protein from the Neisseria gonorrhoeae Mismatch Repair System: Interaction with ATP and DNA].

The mismatch repair system (MMR) ensures the stability of genetic information during DNA replication in almost all organisms. Mismatch repair is initiated after recognition of a non-canonical nucleotide pair by the MutS protein and the formation of a complex between MutS and MutL. Eukaryotic and most bacterial MutL homologs function as endonucleases that introduce a single-strand break in the daughter strand of the DNA, thus activating the repair process. However, many aspects of the functioning of this protein remain unknown. We studied the ATPase and DNA binding functions of the MutL protein from the pathogenic bacterium Neisseria gonorrhoeae (NgoMutL), which exhibits endonuclease activity. For the first time, the kinetic parameters of ATP hydrolysis by the full-length NgoMutL protein were determined. Its interactions with single- and double-stranded DNA fragments of various lengths were studied. NgoMutL was shown to be able to efficiently form complexes with DNA fragments that are longer than 40 nucleotides. Using modified DNA duplexes harboring a 2-pyridyldisulfide group on linkers of various lengths, we obtained NgoMutL conjugates with DNA for the first time. According to these results, the Cys residues of the wild-type protein are located at a distance of approximately 18-50 Šfrom the duplex. The efficiency of the affinity modification of Cys residues in NgoMutL with reactive DNAs was shown to decrease in the presence of ATP or its non-hydrolyzable analog, as well as ZnCl2, in the reaction mixture. We hypothesize that the conserved Cys residues of the C-terminal domain of NgoMutL, which are responsible for the coordination of metal ions in the active center of the protein, are involved in its interaction with DNA. This information may be useful in reconstruction of the main stages of MMR in prokaryotes that are different from γ-proteobacteria, as well as in the search for new targets for drugs against N. gonorrhoeae.

Endonuclease Activity of MutL Protein of the Rhodobacter sphaeroides Mismatch Repair System.

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+; Ni2+ 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.

Spectral-Kinetic Analysis of Recombination Reaction of Heme Centers of bd-Type Quinol Oxidase from Escherichia coli with Carbon Monoxide.

Recombination of the isolated, fully reduced bd-type quinol oxidase from Escherichia coli with carbon monoxide was studied by pulsed absorption spectrophotometry with microsecond time resolution. Analysis of the kinetic phases of recombination was carried out using the global analysis of multiwavelength kinetic data ("Global fitting"). It was found that the unresolved photodissociation of CO is followed by a stepwise (with four phases) recombination with characteristic times (τ) of about 20 µs, 250 µs, 1.1 ms, and 24 ms. The 20-µs phase most likely reflects bimolecular recombination of CO with heme d. Two subsequent kinetic transitions, with τ ~ 250 µs and 1.1 ms, were resolved for the first time. It is assumed that the 250-µs phase is heterogeneous and includes two different processes: recombination of CO with ~7% of heme b595 and transition of heme d from a pentacoordinate to a transient hexacoordinate state in this enzyme population. The 24-ms transition probably reflects a return of heme d to the pentacoordinate state in the same protein fraction. The 1.1-ms phase can be explained by recombination of CO with ~15% of heme b558. Possible models of interaction of CO with different heme centers are discussed.