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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

Zn2+ ions and endonuclease activity of NgoL-CTD.The effect of increasing metal ion (ZnCl2) concentration on endonuclease activity of NgoL-CTD was monitored. It was seen that the enzyme exhibits endonuclease activity in the presence of Mn2+ (lanes 6,7 and 8) and not Zn2+ ions (lanes 2 to 5).
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pone-0013726-g006: Zn2+ ions and endonuclease activity of NgoL-CTD.The effect of increasing metal ion (ZnCl2) concentration on endonuclease activity of NgoL-CTD was monitored. It was seen that the enzyme exhibits endonuclease activity in the presence of Mn2+ (lanes 6,7 and 8) and not Zn2+ ions (lanes 2 to 5).

Mentions: In case of BsuL-CTD, the metal binding motif is defined by the stretch DQHAAQERIKYEY extending from residues. It has been seen that residues E468 and H464 of the metal binding motif (along with residues of Motif IV and V) are involved in stabilizing Zn2+ ions (Figure 5B). In case of BsuL, Zn2+ ions do not facilitate endonucleolysis and Mn2+ appears to be the primary cofactor ion necessary for activity. In the presence of both Mn2+ and Zn2+ BsuL exhibits both nicking and double-stranded cleavage activities. NgoL-CTD did not exhibit endonuclease activity (conversion of closed circular form of a plasmid substrate to open circular form) in the presence of Zn2+ ions even up to a maximal concentration of 20 mM (Figure 6). Unlike BsuL, NgoL could achieve endonucleolysis in the presence of Mg2+ ions also in addition to Mn2+ ([13] and Figure 6). Overall, both the enzymes displayed nicking activity in the presence of Mn2+ and not Zn2+ ions.


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)

Zn2+ ions and endonuclease activity of NgoL-CTD.The effect of increasing metal ion (ZnCl2) concentration on endonuclease activity of NgoL-CTD was monitored. It was seen that the enzyme exhibits endonuclease activity in the presence of Mn2+ (lanes 6,7 and 8) and not Zn2+ ions (lanes 2 to 5).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0013726-g006: Zn2+ ions and endonuclease activity of NgoL-CTD.The effect of increasing metal ion (ZnCl2) concentration on endonuclease activity of NgoL-CTD was monitored. It was seen that the enzyme exhibits endonuclease activity in the presence of Mn2+ (lanes 6,7 and 8) and not Zn2+ ions (lanes 2 to 5).
Mentions: In case of BsuL-CTD, the metal binding motif is defined by the stretch DQHAAQERIKYEY extending from residues. It has been seen that residues E468 and H464 of the metal binding motif (along with residues of Motif IV and V) are involved in stabilizing Zn2+ ions (Figure 5B). In case of BsuL, Zn2+ ions do not facilitate endonucleolysis and Mn2+ appears to be the primary cofactor ion necessary for activity. In the presence of both Mn2+ and Zn2+ BsuL exhibits both nicking and double-stranded cleavage activities. NgoL-CTD did not exhibit endonuclease activity (conversion of closed circular form of a plasmid substrate to open circular form) in the presence of Zn2+ ions even up to a maximal concentration of 20 mM (Figure 6). Unlike BsuL, NgoL could achieve endonucleolysis in the presence of Mg2+ ions also in addition to Mn2+ ([13] and Figure 6). Overall, both the enzymes displayed nicking activity in the presence of Mn2+ and not Zn2+ ions.

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