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

Abstract

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.

Responses of DNA Mismatch Repair Proteins to a Stable G-Quadruplex Embedded into a DNA Duplex Structure

“…These findings are consistent with the CD and UV spectroscopy data that were obtained with shorter 41/22 (24) DNA analogs ( Figure 4) to improve the signal-to-noise ratio. As evident from the CD spectra, the d(GGGT) 4 insert in both single-and double-stranded contexts has the characteristic features of a parallel G4 folding pattern wherein all the guanosines in the G-quartets are in an anti-conformation ( Figure 6).…”

“…Proteins MutS, MutL, and MutH from E. coli as well as MutL from R. sphaeroides were purified as described previously [24,79]. RsMutS was obtained for the first time in this work.…”

“…This method has been described earlier [24]. Briefly, 10 nM pUC-MMR plasmid was incubated with 250 nM rsMutL (per dimer) in 10 mM HEPES-KOH buffer (pH 8.0) containing 100 mM KCl and 5 mM MgCl 2 or MnCl 2 at 37 • C for 1 h. The reaction was terminated by the addition of 50 mM EDTA and 10 activity units of proteinase K with subsequent incubation of the mixture for 30 min at 37 • C. In control experiments, the pUC-MMR plasmid was incubated with R.BamHI (restriction endonuclease) or Nt.Bpu10I (nicking endonuclease) for 60 min at 37 • C in 10 µL of the buffer recommended for each enzyme.…”

“…As for R. sphaeroides, it is a highly adaptive purple bacterium that can survive in various environmental conditions. This property of R. sphaeroides probably has something to do with the potent functioning of repair systems including methyl-independent MMR [24].…”

“…As for R. sphaeroides, it is a highly adaptive purple bacterium that can survive in various environmental conditions. This property of R. sphaeroides probably has something to do with the potent functioning of repair systems including methyl-independent MMR [24]. The main aim of our study was to answer the following questions: whether G4 formation interferes with mismatch-provoked incision of DNA caused by the coordinated actions of proteins MutS, MutL, and MutH, and whether the G4 itself activates MMR responses.…”