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Multiple functions for the N-terminal region of Msh6.

Clark AB, Deterding L, Tomer KB, Kunkel TA - Nucleic Acids Res. (2007)

Bottom Line: Partial proteolysis, DNA affinity chromatography and mass spectrometry identified a fragment comprised of residues 228-299 of yeast Msh6 that binds to DNA and is rich in positively charged residues.Deleting these residues, or replacing lysines and arginines with glutamate, reduces DNA binding in vitro and elevates spontaneous mutation rates and resistance to MNNG treatment in vivo.Similar in vivo defects are conferred by alanine substitutions in a highly conserved motif in the NTR that immediately precedes domain I of MutS proteins, the domain that interacts with mismatched DNA.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.

ABSTRACT
The eukaryotic mismatch repair protein Msh6 shares five domains in common with other MutS members. However, it also contains several hundred additional residues at its N-terminus. A few of these residues bind to PCNA, but the functions of the other amino acids in the N-terminal region (NTR) are unknown. Here we demonstrate that the Msh6 NTR binds to duplex DNA in a salt-sensitive, mismatch-independent manner. Partial proteolysis, DNA affinity chromatography and mass spectrometry identified a fragment comprised of residues 228-299 of yeast Msh6 that binds to DNA and is rich in positively charged residues. Deleting these residues, or replacing lysines and arginines with glutamate, reduces DNA binding in vitro and elevates spontaneous mutation rates and resistance to MNNG treatment in vivo. Similar in vivo defects are conferred by alanine substitutions in a highly conserved motif in the NTR that immediately precedes domain I of MutS proteins, the domain that interacts with mismatched DNA. These data suggest that, in addition to PCNA binding, DNA binding and possibly other functions in the amino terminal region of Msh6 are important for eukaryotic DNA mismatch repair and cellular response to alkylation damage.

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Related in: MedlinePlus

Residues of interest in the yeast and human Msh6 NTRs. The N-terminal 310 amino acids from S. cerevisiae are shown. Acidic residues are colored red and basic residues are colored blue. The green box highlights the PIP box, the red box highlights the region of acidic residues from 144 to 212 (a putative ‘DNA mimic’), the blue box highlights the DNA binding fragment identified by mass spectrometry and the black box outlines residues that are highly conserved in Msh6 homologs. See text for further descriptions.
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Figure 1: Residues of interest in the yeast and human Msh6 NTRs. The N-terminal 310 amino acids from S. cerevisiae are shown. Acidic residues are colored red and basic residues are colored blue. The green box highlights the PIP box, the red box highlights the region of acidic residues from 144 to 212 (a putative ‘DNA mimic’), the blue box highlights the DNA binding fragment identified by mass spectrometry and the black box outlines residues that are highly conserved in Msh6 homologs. See text for further descriptions.

Mentions: In addition to residues important for binding to PCNA, other residues in the NTR, diagramed in Figure 1, could also be functionally important. This importance is suggested by the evolutionary conservation of NTRs in both Msh6 and Msh3 (albeit of different lengths and sequence), by the identification (11) in human Msh6 NTR of a PWWP domain characteristic of proteins associated with chromatin, and by the presence in the human Msh6 NTR of missense mutations that are associated with cancer (12–14). In the present study, we examine the possibility that residues in the Msh6 NTR other than those in the PIP box are functionally important. We demonstrate that recombinant yeast and human Msh6 NTRs bind to duplex DNA, identify amino acids in yeast Msh6 that contribute to this binding, and characterize msh6 mutants that concomitantly reduce DNA binding in vitro and reduce Msh6-dependent mismatch repair and sensitivity to killing by MNNG in vivo. We also show that substituting alanine for residues in a previously unrecognized, highly conserved motif at the extreme C-terminus of the Msh6 NTR also reduce Msh6-dependent mismatch repair and sensitivity to MNNG treatment. These results suggest that the Msh6 NTR has multiple roles in Msh6-dependent mismatch repair and in cellular response to alkylation damage.Figure 1.


Multiple functions for the N-terminal region of Msh6.

Clark AB, Deterding L, Tomer KB, Kunkel TA - Nucleic Acids Res. (2007)

Residues of interest in the yeast and human Msh6 NTRs. The N-terminal 310 amino acids from S. cerevisiae are shown. Acidic residues are colored red and basic residues are colored blue. The green box highlights the PIP box, the red box highlights the region of acidic residues from 144 to 212 (a putative ‘DNA mimic’), the blue box highlights the DNA binding fragment identified by mass spectrometry and the black box outlines residues that are highly conserved in Msh6 homologs. See text for further descriptions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919485&req=5

Figure 1: Residues of interest in the yeast and human Msh6 NTRs. The N-terminal 310 amino acids from S. cerevisiae are shown. Acidic residues are colored red and basic residues are colored blue. The green box highlights the PIP box, the red box highlights the region of acidic residues from 144 to 212 (a putative ‘DNA mimic’), the blue box highlights the DNA binding fragment identified by mass spectrometry and the black box outlines residues that are highly conserved in Msh6 homologs. See text for further descriptions.
Mentions: In addition to residues important for binding to PCNA, other residues in the NTR, diagramed in Figure 1, could also be functionally important. This importance is suggested by the evolutionary conservation of NTRs in both Msh6 and Msh3 (albeit of different lengths and sequence), by the identification (11) in human Msh6 NTR of a PWWP domain characteristic of proteins associated with chromatin, and by the presence in the human Msh6 NTR of missense mutations that are associated with cancer (12–14). In the present study, we examine the possibility that residues in the Msh6 NTR other than those in the PIP box are functionally important. We demonstrate that recombinant yeast and human Msh6 NTRs bind to duplex DNA, identify amino acids in yeast Msh6 that contribute to this binding, and characterize msh6 mutants that concomitantly reduce DNA binding in vitro and reduce Msh6-dependent mismatch repair and sensitivity to killing by MNNG in vivo. We also show that substituting alanine for residues in a previously unrecognized, highly conserved motif at the extreme C-terminus of the Msh6 NTR also reduce Msh6-dependent mismatch repair and sensitivity to MNNG treatment. These results suggest that the Msh6 NTR has multiple roles in Msh6-dependent mismatch repair and in cellular response to alkylation damage.Figure 1.

Bottom Line: Partial proteolysis, DNA affinity chromatography and mass spectrometry identified a fragment comprised of residues 228-299 of yeast Msh6 that binds to DNA and is rich in positively charged residues.Deleting these residues, or replacing lysines and arginines with glutamate, reduces DNA binding in vitro and elevates spontaneous mutation rates and resistance to MNNG treatment in vivo.Similar in vivo defects are conferred by alanine substitutions in a highly conserved motif in the NTR that immediately precedes domain I of MutS proteins, the domain that interacts with mismatched DNA.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.

ABSTRACT
The eukaryotic mismatch repair protein Msh6 shares five domains in common with other MutS members. However, it also contains several hundred additional residues at its N-terminus. A few of these residues bind to PCNA, but the functions of the other amino acids in the N-terminal region (NTR) are unknown. Here we demonstrate that the Msh6 NTR binds to duplex DNA in a salt-sensitive, mismatch-independent manner. Partial proteolysis, DNA affinity chromatography and mass spectrometry identified a fragment comprised of residues 228-299 of yeast Msh6 that binds to DNA and is rich in positively charged residues. Deleting these residues, or replacing lysines and arginines with glutamate, reduces DNA binding in vitro and elevates spontaneous mutation rates and resistance to MNNG treatment in vivo. Similar in vivo defects are conferred by alanine substitutions in a highly conserved motif in the NTR that immediately precedes domain I of MutS proteins, the domain that interacts with mismatched DNA. These data suggest that, in addition to PCNA binding, DNA binding and possibly other functions in the amino terminal region of Msh6 are important for eukaryotic DNA mismatch repair and cellular response to alkylation damage.

Show MeSH
Related in: MedlinePlus