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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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DNA binding by yeast and human Msh6 NTRs. A. Binding of the yeast NTR (left panel) and human NTR (right panel) to dsDNA cellulose. Purified NTR protein (50 µg) was diluted in 1 ml of 20 mM Tris pH 8, 15 mM NaCl, 1 mM EDTA, 1 mM β-mercaptoethanol, 10% glycerol (column buffer) and applied to a 0.1-ml volume of dsDNA cellulose (Sigma) packed into Poly-Prep chromatography columns (Bio-Rad). Columns were washed with 1 ml of column buffer and step eluted with 0.2 ml of column buffer containing NaCl concentrations from 25 to 300 mM. B. Nitrocellulose filter binding assays were performed as described in Materials and Methods. Twenty-microliter reactions containing between 2 fmoles and 2 pmoles of purified yeast N-terminal 299 (filled circle) or human N-terminal 394 (open square) residues were incubated with 1 nmole of nucleotide of 3H labeled pGBT9. DNA bound to protein is retained during filtration through nitrocellulose membranes. For comparison, pGBT9 binding by identical concentrations of yMutSα is shown (filled square). Binding of yN299 to 1 nmole of 3H labeled M13mp2 phage DNA (open circle) shows reduced affinity for ssDNA. C. The binding of 0.3 to 100 pmol of either the yeast N-terminal 299 residues (open symbols) and human N-terminal 394 residues (closed symbols) to 0.1 pmole of heteroduplex (G·T41), homoduplex (A·T41) or ssDNA oligonucleotide substrates in 20 µl reactions was monitored by electrophoretic mobility shift assays as described in Materials and Methods. The fraction of substrate bound is the ratio of substrate migrating with a slower mobility compared with the total amount of substrate in the lane in comparison with a reference mock lane lacking NTR protein.
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Figure 2: DNA binding by yeast and human Msh6 NTRs. A. Binding of the yeast NTR (left panel) and human NTR (right panel) to dsDNA cellulose. Purified NTR protein (50 µg) was diluted in 1 ml of 20 mM Tris pH 8, 15 mM NaCl, 1 mM EDTA, 1 mM β-mercaptoethanol, 10% glycerol (column buffer) and applied to a 0.1-ml volume of dsDNA cellulose (Sigma) packed into Poly-Prep chromatography columns (Bio-Rad). Columns were washed with 1 ml of column buffer and step eluted with 0.2 ml of column buffer containing NaCl concentrations from 25 to 300 mM. B. Nitrocellulose filter binding assays were performed as described in Materials and Methods. Twenty-microliter reactions containing between 2 fmoles and 2 pmoles of purified yeast N-terminal 299 (filled circle) or human N-terminal 394 (open square) residues were incubated with 1 nmole of nucleotide of 3H labeled pGBT9. DNA bound to protein is retained during filtration through nitrocellulose membranes. For comparison, pGBT9 binding by identical concentrations of yMutSα is shown (filled square). Binding of yN299 to 1 nmole of 3H labeled M13mp2 phage DNA (open circle) shows reduced affinity for ssDNA. C. The binding of 0.3 to 100 pmol of either the yeast N-terminal 299 residues (open symbols) and human N-terminal 394 residues (closed symbols) to 0.1 pmole of heteroduplex (G·T41), homoduplex (A·T41) or ssDNA oligonucleotide substrates in 20 µl reactions was monitored by electrophoretic mobility shift assays as described in Materials and Methods. The fraction of substrate bound is the ratio of substrate migrating with a slower mobility compared with the total amount of substrate in the lane in comparison with a reference mock lane lacking NTR protein.

Mentions: When the structure of Thermus aquaticus MutS protein was solved, a structure-based amino acid sequence alignment was provided [Figure 5 in (6)]. This alignment suggested that domain I of Msh6 may begin at approximately residue 300 in yeast Msh6 and at approximately residue 395 in human MSH6. On that basis, and absent structural information on Msh6 proteins per se, here we studied and refer to the preceding residues as Msh6 NTRs. As a first step towards determining if these Msh6 NTRs interact with macromolecules other than PCNA, we expressed and purified the yeast Msh6 NTR comprised of residues 1–299 and the human Msh6 NTR comprised of residues 1–394. Both proteins were expressed in E. coli with a 6-His tag. This tag was placed at the N-terminus of the yeast NTR, but at the C-terminus of the human NTR to avoid perturbing the PIP box that is at the extreme N-terminus. Both NTRs were purified using three chromatographic steps, one of which involved binding to a heparin column. Both NTRs were obtained in highly purified form (see lanes labeled ‘load’ in Figure 2A). Because heparin is a negatively charged resin to which many DNA binding proteins bind, we tested whether the Msh6 NTRs could bind to a dsDNA cellulose column. Indeed, the NTR of yeast Msh6 bound, and peak fractions eluted from the column at 125–150 mM NaCl (Figure 2A). The NTR of human Msh6 also bound, and the peak fraction eluted from the column at 225 mM NaCl (Figure 2A). Thus both proteins can bind to dsDNA via ionic interactions, and the NTR of human Msh6 appears to bind more tightly than the NTR of yeast Msh6. When DNA binding capacity was measured using a filter-binding assay (16), both the yeast and human NTRs bound to double-stranded plasmid DNA with an affinity similar to yMutSα (Figure 2B) The yMsh6 NTR also bound to single-stranded M13 DNA (open circles in Figure 2B), but with lower affinity. Using an electrophoretic mobility shift assay (EMSA), both yeast and human Msh6 NTRs were observed to bind similarly to homoduplex DNA and to heteroduplex DNA containing a G–T mismatch (Figure 2C).Figure 2.


Multiple functions for the N-terminal region of Msh6.

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

DNA binding by yeast and human Msh6 NTRs. A. Binding of the yeast NTR (left panel) and human NTR (right panel) to dsDNA cellulose. Purified NTR protein (50 µg) was diluted in 1 ml of 20 mM Tris pH 8, 15 mM NaCl, 1 mM EDTA, 1 mM β-mercaptoethanol, 10% glycerol (column buffer) and applied to a 0.1-ml volume of dsDNA cellulose (Sigma) packed into Poly-Prep chromatography columns (Bio-Rad). Columns were washed with 1 ml of column buffer and step eluted with 0.2 ml of column buffer containing NaCl concentrations from 25 to 300 mM. B. Nitrocellulose filter binding assays were performed as described in Materials and Methods. Twenty-microliter reactions containing between 2 fmoles and 2 pmoles of purified yeast N-terminal 299 (filled circle) or human N-terminal 394 (open square) residues were incubated with 1 nmole of nucleotide of 3H labeled pGBT9. DNA bound to protein is retained during filtration through nitrocellulose membranes. For comparison, pGBT9 binding by identical concentrations of yMutSα is shown (filled square). Binding of yN299 to 1 nmole of 3H labeled M13mp2 phage DNA (open circle) shows reduced affinity for ssDNA. C. The binding of 0.3 to 100 pmol of either the yeast N-terminal 299 residues (open symbols) and human N-terminal 394 residues (closed symbols) to 0.1 pmole of heteroduplex (G·T41), homoduplex (A·T41) or ssDNA oligonucleotide substrates in 20 µl reactions was monitored by electrophoretic mobility shift assays as described in Materials and Methods. The fraction of substrate bound is the ratio of substrate migrating with a slower mobility compared with the total amount of substrate in the lane in comparison with a reference mock lane lacking NTR protein.
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Figure 2: DNA binding by yeast and human Msh6 NTRs. A. Binding of the yeast NTR (left panel) and human NTR (right panel) to dsDNA cellulose. Purified NTR protein (50 µg) was diluted in 1 ml of 20 mM Tris pH 8, 15 mM NaCl, 1 mM EDTA, 1 mM β-mercaptoethanol, 10% glycerol (column buffer) and applied to a 0.1-ml volume of dsDNA cellulose (Sigma) packed into Poly-Prep chromatography columns (Bio-Rad). Columns were washed with 1 ml of column buffer and step eluted with 0.2 ml of column buffer containing NaCl concentrations from 25 to 300 mM. B. Nitrocellulose filter binding assays were performed as described in Materials and Methods. Twenty-microliter reactions containing between 2 fmoles and 2 pmoles of purified yeast N-terminal 299 (filled circle) or human N-terminal 394 (open square) residues were incubated with 1 nmole of nucleotide of 3H labeled pGBT9. DNA bound to protein is retained during filtration through nitrocellulose membranes. For comparison, pGBT9 binding by identical concentrations of yMutSα is shown (filled square). Binding of yN299 to 1 nmole of 3H labeled M13mp2 phage DNA (open circle) shows reduced affinity for ssDNA. C. The binding of 0.3 to 100 pmol of either the yeast N-terminal 299 residues (open symbols) and human N-terminal 394 residues (closed symbols) to 0.1 pmole of heteroduplex (G·T41), homoduplex (A·T41) or ssDNA oligonucleotide substrates in 20 µl reactions was monitored by electrophoretic mobility shift assays as described in Materials and Methods. The fraction of substrate bound is the ratio of substrate migrating with a slower mobility compared with the total amount of substrate in the lane in comparison with a reference mock lane lacking NTR protein.
Mentions: When the structure of Thermus aquaticus MutS protein was solved, a structure-based amino acid sequence alignment was provided [Figure 5 in (6)]. This alignment suggested that domain I of Msh6 may begin at approximately residue 300 in yeast Msh6 and at approximately residue 395 in human MSH6. On that basis, and absent structural information on Msh6 proteins per se, here we studied and refer to the preceding residues as Msh6 NTRs. As a first step towards determining if these Msh6 NTRs interact with macromolecules other than PCNA, we expressed and purified the yeast Msh6 NTR comprised of residues 1–299 and the human Msh6 NTR comprised of residues 1–394. Both proteins were expressed in E. coli with a 6-His tag. This tag was placed at the N-terminus of the yeast NTR, but at the C-terminus of the human NTR to avoid perturbing the PIP box that is at the extreme N-terminus. Both NTRs were purified using three chromatographic steps, one of which involved binding to a heparin column. Both NTRs were obtained in highly purified form (see lanes labeled ‘load’ in Figure 2A). Because heparin is a negatively charged resin to which many DNA binding proteins bind, we tested whether the Msh6 NTRs could bind to a dsDNA cellulose column. Indeed, the NTR of yeast Msh6 bound, and peak fractions eluted from the column at 125–150 mM NaCl (Figure 2A). The NTR of human Msh6 also bound, and the peak fraction eluted from the column at 225 mM NaCl (Figure 2A). Thus both proteins can bind to dsDNA via ionic interactions, and the NTR of human Msh6 appears to bind more tightly than the NTR of yeast Msh6. When DNA binding capacity was measured using a filter-binding assay (16), both the yeast and human NTRs bound to double-stranded plasmid DNA with an affinity similar to yMutSα (Figure 2B) The yMsh6 NTR also bound to single-stranded M13 DNA (open circles in Figure 2B), but with lower affinity. Using an electrophoretic mobility shift assay (EMSA), both yeast and human Msh6 NTRs were observed to bind similarly to homoduplex DNA and to heteroduplex DNA containing a G–T mismatch (Figure 2C).Figure 2.

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