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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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Characterization of NgoL-CTD-L480E-L487E.The change in oligomeric status of the mutant protein was characterized by assessing differences in the products of chemical crosslinking and CD spectra. (A) Chemical crosslinking of NgoL-CTD wild type and NgoL-CTD- L480E-L487E using glutaraldehyde: The NgoL-CTD wild type and NgoL-CTD- L480E-L487E (10 μM) were treated with increasing concentrations of 0.05% glutaraldehyde and products analyzed as described in Materials and methods section. Lane 1, NgoL-CTD-L480E-L487E alone; lane 2, 3 & 4, NgoL-CTD-L480E-L487E and increasing concentration of glutaraldehyde; lane 5, 6 & 7, NgoL-CTD wild type and increasing concentration of glutaraldehyde, lane 8, NgoL-CTD wild type alone; M, molecular weight markers. (B). CD spectra of NgoL-CTD and NgoL-CTD-L480E-L487E. Both the proteins were dialyzed against 10 mM potassium phosphate (pH 8) buffer containing 30 mM KCl. CD spectra were recorded from 200 to 300 nm as described in Materials and methods.
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pone-0013726-g011: Characterization of NgoL-CTD-L480E-L487E.The change in oligomeric status of the mutant protein was characterized by assessing differences in the products of chemical crosslinking and CD spectra. (A) Chemical crosslinking of NgoL-CTD wild type and NgoL-CTD- L480E-L487E using glutaraldehyde: The NgoL-CTD wild type and NgoL-CTD- L480E-L487E (10 μM) were treated with increasing concentrations of 0.05% glutaraldehyde and products analyzed as described in Materials and methods section. Lane 1, NgoL-CTD-L480E-L487E alone; lane 2, 3 & 4, NgoL-CTD-L480E-L487E and increasing concentration of glutaraldehyde; lane 5, 6 & 7, NgoL-CTD wild type and increasing concentration of glutaraldehyde, lane 8, NgoL-CTD wild type alone; M, molecular weight markers. (B). CD spectra of NgoL-CTD and NgoL-CTD-L480E-L487E. Both the proteins were dialyzed against 10 mM potassium phosphate (pH 8) buffer containing 30 mM KCl. CD spectra were recorded from 200 to 300 nm as described in Materials and methods.

Mentions: In ordered to get additional information about the oligomeric status, we employed chemical cross-linking of the protein by glutaraldehyde. Glutaraldehyde is a homobifunctional cross-linking reagent that cross-links N-terminal primary amines, resulting in the formation of Schiff's base. Both NgoL-CTD and NgoL-CTD-L480E-L487E were subjected to glutaraldehyde treatment followed by SDS-PAGE analysis. Analysis of the cross-linking results revealed that the wild type formed significant amount of crosslinked product – corresponding to dimer of NgoL-CTD- that increased as a function of glutaraldehyde concentration (Figure 11A). In case of NgoL-CTD-L480E-L487E no crosslinked products were observed and a single band corresponding to a molecular mass of 27 kDa was observed. These results indicate that, the two mutations L480E and L487E completely destabilized the dimer.


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)

Characterization of NgoL-CTD-L480E-L487E.The change in oligomeric status of the mutant protein was characterized by assessing differences in the products of chemical crosslinking and CD spectra. (A) Chemical crosslinking of NgoL-CTD wild type and NgoL-CTD- L480E-L487E using glutaraldehyde: The NgoL-CTD wild type and NgoL-CTD- L480E-L487E (10 μM) were treated with increasing concentrations of 0.05% glutaraldehyde and products analyzed as described in Materials and methods section. Lane 1, NgoL-CTD-L480E-L487E alone; lane 2, 3 & 4, NgoL-CTD-L480E-L487E and increasing concentration of glutaraldehyde; lane 5, 6 & 7, NgoL-CTD wild type and increasing concentration of glutaraldehyde, lane 8, NgoL-CTD wild type alone; M, molecular weight markers. (B). CD spectra of NgoL-CTD and NgoL-CTD-L480E-L487E. Both the proteins were dialyzed against 10 mM potassium phosphate (pH 8) buffer containing 30 mM KCl. CD spectra were recorded from 200 to 300 nm as described in Materials and methods.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0013726-g011: Characterization of NgoL-CTD-L480E-L487E.The change in oligomeric status of the mutant protein was characterized by assessing differences in the products of chemical crosslinking and CD spectra. (A) Chemical crosslinking of NgoL-CTD wild type and NgoL-CTD- L480E-L487E using glutaraldehyde: The NgoL-CTD wild type and NgoL-CTD- L480E-L487E (10 μM) were treated with increasing concentrations of 0.05% glutaraldehyde and products analyzed as described in Materials and methods section. Lane 1, NgoL-CTD-L480E-L487E alone; lane 2, 3 & 4, NgoL-CTD-L480E-L487E and increasing concentration of glutaraldehyde; lane 5, 6 & 7, NgoL-CTD wild type and increasing concentration of glutaraldehyde, lane 8, NgoL-CTD wild type alone; M, molecular weight markers. (B). CD spectra of NgoL-CTD and NgoL-CTD-L480E-L487E. Both the proteins were dialyzed against 10 mM potassium phosphate (pH 8) buffer containing 30 mM KCl. CD spectra were recorded from 200 to 300 nm as described in Materials and methods.
Mentions: In ordered to get additional information about the oligomeric status, we employed chemical cross-linking of the protein by glutaraldehyde. Glutaraldehyde is a homobifunctional cross-linking reagent that cross-links N-terminal primary amines, resulting in the formation of Schiff's base. Both NgoL-CTD and NgoL-CTD-L480E-L487E were subjected to glutaraldehyde treatment followed by SDS-PAGE analysis. Analysis of the cross-linking results revealed that the wild type formed significant amount of crosslinked product – corresponding to dimer of NgoL-CTD- that increased as a function of glutaraldehyde concentration (Figure 11A). In case of NgoL-CTD-L480E-L487E no crosslinked products were observed and a single band corresponding to a molecular mass of 27 kDa was observed. These results indicate that, the two mutations L480E and L487E completely destabilized the dimer.

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