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The C-terminal domain of the MutL homolog from Neisseria gonorrhoeae forms an inverted homodimer.

Namadurai S, Jain D, Kulkarni DS, Tabib CR, Friedhoff P, Rao DN, Nair DT - PLoS ONE (2010)

Bottom Line: Many bacteria and eukaryotes, including humans, do not possess a homolog of MutH.Such an arrangement raises the possibility that one of the active sites is occluded due to interaction of NgoL with other protein factors involved in MMR.The presentation of only one active site to substrate DNA will ensure that nicking of only one strand occurs to prevent inadvertent and deleterious double stranded cleavage.

View Article: PubMed Central - PubMed

Affiliation: Laboratory 4, National Centre for Biological Sciences, Bangalore, India.

ABSTRACT
The mismatch repair (MMR) pathway serves to maintain the integrity of the genome by removing mispaired bases from the newly synthesized strand. In E. coli, MutS, MutL and MutH coordinate to discriminate the daughter strand through a mechanism involving lack of methylation on the new strand. This facilitates the creation of a nick by MutH in the daughter strand to initiate mismatch repair. Many bacteria and eukaryotes, including humans, do not possess a homolog of MutH. Although the exact strategy for strand discrimination in these organisms is yet to be ascertained, the required nicking endonuclease activity is resident in the C-terminal domain of MutL. This activity is dependent on the integrity of a conserved metal binding motif. Unlike their eukaryotic counterparts, MutL in bacteria like Neisseria exist in the form of a homodimer. Even though this homodimer would possess two active sites, it still acts a nicking endonuclease. Here, we present the crystal structure of the C-terminal domain (CTD) of the MutL homolog of Neisseria gonorrhoeae (NgoL) determined to a resolution of 2.4 Å. The structure shows that the metal binding motif exists in a helical configuration and that four of the six conserved motifs in the MutL family, including the metal binding site, localize together to form a composite active site. NgoL-CTD exists in the form of an elongated inverted homodimer stabilized by a hydrophobic interface rich in leucines. The inverted arrangement places the two composite active sites in each subunit on opposite lateral sides of the homodimer. Such an arrangement raises the possibility that one of the active sites is occluded due to interaction of NgoL with other protein factors involved in MMR. The presentation of only one active site to substrate DNA will ensure that nicking of only one strand occurs to prevent inadvertent and deleterious double stranded cleavage.

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Related in: MedlinePlus

Electron Density Map.A section of a composite Omit Map is displayed at a contour level of 1. (δ = 1.0). Electron density corresponding to the metal binding motif is displayed. The metal binding motif is shown in stick representation and colored according to element.
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pone-0013726-g001: Electron Density Map.A section of a composite Omit Map is displayed at a contour level of 1. (δ = 1.0). Electron density corresponding to the metal binding motif is displayed. The metal binding motif is shown in stick representation and colored according to element.

Mentions: The (NgoL-CTD) crystals belong to space group P21 with cell dimensions of a = 49.5 Å, b = 62.1 Å, c = 92.1 Å and α = γ = 90°, β = 104.6°. The structure was determined by single-wavelength anomalous diffraction (SAD) method using selenomethionine-labeled NgoL-CTD. An experimental electron density map of good quality was obtained and was used to build the model. The structure has been refined to a resolution of 2.4 Å with a final Rfree and Rcrys of 27.3% and 22.7%, respectively and the quality of the final model was confirmed through a composite omit map (Figure 1). The final NgoL-CTD structure includes two monomers. For monomer A, residues 463–654 and for monomer B, residues 464–656 could be built and there is no density for five residues extending from 587–591 in both monomers. The structure also shows the presence of 108 water molecules and exhibits good stereochemistry, with 96% of the residues in the favored regions of the Ramachandran plot.


The C-terminal domain of the MutL homolog from Neisseria gonorrhoeae forms an inverted homodimer.

Namadurai S, Jain D, Kulkarni DS, Tabib CR, Friedhoff P, Rao DN, Nair DT - PLoS ONE (2010)

Electron Density Map.A section of a composite Omit Map is displayed at a contour level of 1. (δ = 1.0). Electron density corresponding to the metal binding motif is displayed. The metal binding motif is shown in stick representation and colored according to element.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2965676&req=5

pone-0013726-g001: Electron Density Map.A section of a composite Omit Map is displayed at a contour level of 1. (δ = 1.0). Electron density corresponding to the metal binding motif is displayed. The metal binding motif is shown in stick representation and colored according to element.
Mentions: The (NgoL-CTD) crystals belong to space group P21 with cell dimensions of a = 49.5 Å, b = 62.1 Å, c = 92.1 Å and α = γ = 90°, β = 104.6°. The structure was determined by single-wavelength anomalous diffraction (SAD) method using selenomethionine-labeled NgoL-CTD. An experimental electron density map of good quality was obtained and was used to build the model. The structure has been refined to a resolution of 2.4 Å with a final Rfree and Rcrys of 27.3% and 22.7%, respectively and the quality of the final model was confirmed through a composite omit map (Figure 1). The final NgoL-CTD structure includes two monomers. For monomer A, residues 463–654 and for monomer B, residues 464–656 could be built and there is no density for five residues extending from 587–591 in both monomers. The structure also shows the presence of 108 water molecules and exhibits good stereochemistry, with 96% of the residues in the favored regions of the Ramachandran plot.

Bottom Line: Many bacteria and eukaryotes, including humans, do not possess a homolog of MutH.Such an arrangement raises the possibility that one of the active sites is occluded due to interaction of NgoL with other protein factors involved in MMR.The presentation of only one active site to substrate DNA will ensure that nicking of only one strand occurs to prevent inadvertent and deleterious double stranded cleavage.

View Article: PubMed Central - PubMed

Affiliation: Laboratory 4, National Centre for Biological Sciences, Bangalore, India.

ABSTRACT
The mismatch repair (MMR) pathway serves to maintain the integrity of the genome by removing mispaired bases from the newly synthesized strand. In E. coli, MutS, MutL and MutH coordinate to discriminate the daughter strand through a mechanism involving lack of methylation on the new strand. This facilitates the creation of a nick by MutH in the daughter strand to initiate mismatch repair. Many bacteria and eukaryotes, including humans, do not possess a homolog of MutH. Although the exact strategy for strand discrimination in these organisms is yet to be ascertained, the required nicking endonuclease activity is resident in the C-terminal domain of MutL. This activity is dependent on the integrity of a conserved metal binding motif. Unlike their eukaryotic counterparts, MutL in bacteria like Neisseria exist in the form of a homodimer. Even though this homodimer would possess two active sites, it still acts a nicking endonuclease. Here, we present the crystal structure of the C-terminal domain (CTD) of the MutL homolog of Neisseria gonorrhoeae (NgoL) determined to a resolution of 2.4 Å. The structure shows that the metal binding motif exists in a helical configuration and that four of the six conserved motifs in the MutL family, including the metal binding site, localize together to form a composite active site. NgoL-CTD exists in the form of an elongated inverted homodimer stabilized by a hydrophobic interface rich in leucines. The inverted arrangement places the two composite active sites in each subunit on opposite lateral sides of the homodimer. Such an arrangement raises the possibility that one of the active sites is occluded due to interaction of NgoL with other protein factors involved in MMR. The presentation of only one active site to substrate DNA will ensure that nicking of only one strand occurs to prevent inadvertent and deleterious double stranded cleavage.

Show MeSH
Related in: MedlinePlus