Pseudomonas aeruginosa MutL protein functions in Escherichia coli

Abstract

Escherichia coli MutS, MutL and MutH proteins act sequentially in the MMRS (mismatch repair system). MutH directs the repair system to the newly synthesized strand due to its transient lack of Dam (DNA-adenine methylase) methylation. Although Pseudomonas aeruginosa does not have the corresponding E. coli MutH and Dam homologues, and consequently the MMRS seems to work differently, we show that the mutL gene from P. aeruginosa is capable of complementing a MutL-deficient strain of E. coli. MutL from P. aeruginosa has conserved 21 out of the 22 amino acids known to affect functioning of E. coli MutL. We showed, using protein affinity chromatography, that the C-terminal regions of P. aeruginosa and E. coli MutL are capable of specifically interacting with E. coli MutH and retaining the E. coli MutH. Although, the amino acid sequences of the C-terminal regions of these two proteins are only 18% identical, they are 88% identical in the predicted secondary structure. Finally, by analysing (E. coli-P. aeruginosa) chimaeric MutL proteins, we show that the N-terminal regions of E. coli and P. aeruginosa MutL proteins function similarly, in vivo and in vitro. These new findings support the hypothesis that a large surface, rather than a single amino acid, constitutes the MutL surface for interaction with MutH, and that the N- and C-terminal regions of MutL are involved in such interactions.

Figure 1. BLAST analysis of bacterial sequenced genomes

BLAST search results using E. coli MutH, Dam, MutS, MutL and UvrD proteins as query for search of homologous proteins within completely sequenced bacterial genomes. Only the highest homologues to E. coli proteins for each bacterial species are indicated. Black vertical lines indicate bacterial species that do not have the MutH/Dam system and the first species within this group is underlined. For each protein the third column indicates the score (bits).

Figure 2. Analysis of adenine methylation within d(GATC) sequences

Plasmids purified from E. coli or P. aeruginosa wild-type strains were digested with MboI or Sau3AI restriction enzymes, which are sensitive and insensitive to adenine methylation respectively, and then separated on 0.8% agarose gel. DNA was visualized by ethidium bromide staining. The negative of the photograph is shown. C, control plasmid from E. coli or P. aeruginosa not digested.

Figure 3. E. coli and P. aeruginosa MutL amino acid sequence comparison

Amino acid sequences were aligned using CLUSTAL W. Amino acids for which mutation in E. coli MutL results in non-functional protein are indicated with grey boxes. Amino acids that are identical (*), strongly similar (:), weakly similar (.) or different () are indicated. Predicted secondary structures are shown between the amino acid sequences. H, helix; E, β-sheet. Black line indicates an unstructured linker region between the N- and C-terminal regions.

Figure 4. In vitro activation of E. coli MutH endonuclease activity

(A) Unmethylated d(GATC) plasmid was digested with increasing amounts of E. coli MutH protein (see the Experimental section), separated on 0.6% agarose gel and visualized with ethidium bromide. S, supercoiled plasmid; Nc, nicked circular plasmid. (B, C, E and F) Unmethylated d(GATC) plasmid was incubated with buffer (–), with 70 nM E. coli MutH (H), with 500 nM of the indicated MutL protein (L) or with 70 nM E. coli MutH and increasing amounts of the indicated MutL protein (H/MutL) (see the Experimental section), before separation on 0.6% agarose gel. (D) Schematic representation of E. coli (Lc), P. aeruginosa (Lp) and chimaeric MutL proteins containing the E. coli N-terminal region and P. aeruginosa C-terminal region (Lc–Lp) or vice versa (Lp–Lc). ApaLI and EcoRI indicate positions of the corresponding restriction sites used for the construction of chimaeric mutL genes. (G) Ethidium bromide-stained gels (B, C, E and F) were scanned and the amount of Nc plasmid was plotted as a percentage. The amount of Nc plasmid obtained with MutH alone (70 nM) was defined as 0%. The difference between the total amount of plasmid used and the amount of Nc plasmid obtained with MutH alone is taken as 100%. Negatives of ethidium bromide photographs are shown.

Figure 5. Analysis of MutL–MutH interaction on protein affinity columns

(A–G) Intein-tagged proteins were adsorbed on to chitin beads and used as affinity ligands. Different His-tagged proteins were loaded on to each column and then eluted with increasing amounts of NaCl, as indicated (see the Experimental section). Gels shown in (A–G) were scanned and the values plotted in (H) (see the Experimental section). Column, protein adsorbed on to chitin beads. P.L., protein loaded on to the column. I, Intein-tagged proteins; His, His-tagged proteins; H, E. coli MutH protein; CLc and CLp, MutL C-terminal regions of E. coli and P. aeruginosa; NLc and NLp, N-terminal regions of E. coli and P. aeruginosa.

Figure 6. Identification of putative amino acids involved in MutL–MutH interaction

Selected docking models for interaction of E. coli LN40(dimer)–MutH [12] were analysed using the RasMol program (see the Experimental section). MutL amino acids in which the C-α is not more than 6 Å from any C-α of MutH are indicated. The number of black triangles indicates how many times each amino acid was identified by this analysis. The four amino acids identified on E. coli MutL protein by cross-linking (Asn¹⁶⁹, Ala²⁵¹, Gln³¹⁴ and Leu³²⁷) [12] are indicated with grey boxes. Amino acids that are identical (*), strongly similar (:), wealhy similar (.) or different () are indicated. Predicted secondary structures are shown between the amino acid sequences. H, helix; E, β-sheet.