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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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Conserved motifs in the MutL family and the NgoL-CTD structure.The position of the six conserved motifs in the MutL family are displayed in the primary, tertiary and quaternary structures of NgoL-CTD. (A) Sequence alignment of representative sequences corresponding to the CTD regions of homologs of MutL. The stretches corresponding to different secondary structural elements are labeled and the conserved motifs I, II, III, IV, V and VI are shaded red, green, teal, magenta, blue and yellow. (B) The location of the conserved sequence motifs in the NgoL-CTD structure is displayed. The residues of the motifs are displayed in stick representation with the same coloring scheme as before.
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pone-0013726-g004: Conserved motifs in the MutL family and the NgoL-CTD structure.The position of the six conserved motifs in the MutL family are displayed in the primary, tertiary and quaternary structures of NgoL-CTD. (A) Sequence alignment of representative sequences corresponding to the CTD regions of homologs of MutL. The stretches corresponding to different secondary structural elements are labeled and the conserved motifs I, II, III, IV, V and VI are shaded red, green, teal, magenta, blue and yellow. (B) The location of the conserved sequence motifs in the NgoL-CTD structure is displayed. The residues of the motifs are displayed in stick representation with the same coloring scheme as before.

Mentions: On the basis of sequence analysis of the MutL family Kosinski et al., had highlighted the presence of six motifs that are conserved to varying degrees [15] (Figure 4A). These consensus sequences map to different regions of the NgoL-CTD structure (Figure 4B). Motif I is highly conserved to a large degree and has the consensus sequence (A/G)Q. It is present in proximal sub-domain (A472-Q473) of NgoL-CTD towards the C-terminus of the β1 strand that is part of the B1 β-sheet involved in dimerization. Motif II has the consensus sequence DQHA(X)2E(X)4E and represents the metal binding motif (DMHAAAERVNYE in NgoL extending from residues 491 to 502) that is critical for catalysis. In a number of orthologs, including BsuL, the residue following the first Glu residue in this motif is a basic residue (Lys, Arg) and this implies that salt bridge formed between R498 and E502 will be a conserved feature of this motif (equivalent residues in BsuL are R468 and E473).


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)

Conserved motifs in the MutL family and the NgoL-CTD structure.The position of the six conserved motifs in the MutL family are displayed in the primary, tertiary and quaternary structures of NgoL-CTD. (A) Sequence alignment of representative sequences corresponding to the CTD regions of homologs of MutL. The stretches corresponding to different secondary structural elements are labeled and the conserved motifs I, II, III, IV, V and VI are shaded red, green, teal, magenta, blue and yellow. (B) The location of the conserved sequence motifs in the NgoL-CTD structure is displayed. The residues of the motifs are displayed in stick representation with the same coloring scheme as before.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0013726-g004: Conserved motifs in the MutL family and the NgoL-CTD structure.The position of the six conserved motifs in the MutL family are displayed in the primary, tertiary and quaternary structures of NgoL-CTD. (A) Sequence alignment of representative sequences corresponding to the CTD regions of homologs of MutL. The stretches corresponding to different secondary structural elements are labeled and the conserved motifs I, II, III, IV, V and VI are shaded red, green, teal, magenta, blue and yellow. (B) The location of the conserved sequence motifs in the NgoL-CTD structure is displayed. The residues of the motifs are displayed in stick representation with the same coloring scheme as before.
Mentions: On the basis of sequence analysis of the MutL family Kosinski et al., had highlighted the presence of six motifs that are conserved to varying degrees [15] (Figure 4A). These consensus sequences map to different regions of the NgoL-CTD structure (Figure 4B). Motif I is highly conserved to a large degree and has the consensus sequence (A/G)Q. It is present in proximal sub-domain (A472-Q473) of NgoL-CTD towards the C-terminus of the β1 strand that is part of the B1 β-sheet involved in dimerization. Motif II has the consensus sequence DQHA(X)2E(X)4E and represents the metal binding motif (DMHAAAERVNYE in NgoL extending from residues 491 to 502) that is critical for catalysis. In a number of orthologs, including BsuL, the residue following the first Glu residue in this motif is a basic residue (Lys, Arg) and this implies that salt bridge formed between R498 and E502 will be a conserved feature of this motif (equivalent residues in BsuL are R468 and E473).

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