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. 2010 Apr 4;9(4):448-57.
doi: 10.1016/j.dnarep.2010.01.010.

Functional residues on the surface of the N-terminal domain of yeast Pms1

Mercedes E Arana  1 , Shannon F Holmes, John M Fortune, Andrea F Moon, Lars C Pedersen, Thomas A Kunkel
Affiliations

Functional residues on the surface of the N-terminal domain of yeast Pms1

Mercedes E Arana et al. DNA Repair (Amst). .

Abstract

Saccharomyces cerevisiae MutLalpha is a heterodimer of Mlh1 and Pms1 that participates in DNA mismatch repair (MMR). Both proteins have weakly conserved C-terminal regions (CTDs), with the CTD of Pms1 harboring an essential endonuclease activity. These proteins also have conserved N-terminal domains (NTDs) that bind and hydrolyze ATP and bind to DNA. To better understand Pms1 functions and potential interactions with DNA and/or other proteins, we solved the 2.5A crystal structure of yeast Pms1 (yPms1) NTD. The structure is similar to the homologous NTDs of Escherichia coli MutL and human PMS2, including the site involved in ATP binding and hydrolysis. The structure reveals a number of conserved, positively charged surface residues that do not interact with other residues in the NTD and are therefore candidates for interactions with DNA, with the CTD and/or with other proteins. When these were replaced with glutamate, several replacements resulted in yeast strains with elevated mutation rates. Two replacements also resulted in NTDs with decreased DNA binding affinity in vitro, suggesting that these residues contribute to DNA binding that is important for mismatch repair. Elevated mutation rates also resulted from surface residue replacements that did not affect DNA binding, suggesting that these conserved residues serve other functions, possibly involving interactions with other MMR proteins.

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Conflict of interest statement

Conflict of Interest Statement

The authors declare that there is no conflict of interest.

Figures

Figure 1
Figure 1. MutL homologs
A. Schematic diagram of E. coli MutL and S.cerevisiae Mlh1 and Pms1 proteins. Diagram depicts the location of the MutL ATP binding domain (yellow) and the location of the Mlh1 and Pms1 ATP binding domains by homology to MutL (yellow) in the highly conserved N-terminal domain. The conserved ATP binding motifs I, II, III, and IV are also represented (blue bars in N-terminal domain) [42,63]. The endonuclease active site at the C-terminal end of Pms1 is depicted (blue bar) [39]. The Pms1 interaction region in Mlh1 and the Mlh1 interaction region in the less well-conserved C-terminal domain of Pms1 are also represented (red) [26]. Figure was adapted from [23]. B. Structural alignment of the N-terminal domains of yPms1 and hPMS2. Secondary structure elements are labeled according to that of hPMS2 [22]. Conserved residues between the two proteins are starred and the lid region is underlined in red. Regions with structural differences have tan background. Residues in orange are disordered, residues in pink were mutated in this study, and side chains for the residues colored blue interact with the bound nucleotide. Individual residues that were mutated and are conserved in yMlh1 are underlined in pink.
Figure 1
Figure 1. MutL homologs
A. Schematic diagram of E. coli MutL and S.cerevisiae Mlh1 and Pms1 proteins. Diagram depicts the location of the MutL ATP binding domain (yellow) and the location of the Mlh1 and Pms1 ATP binding domains by homology to MutL (yellow) in the highly conserved N-terminal domain. The conserved ATP binding motifs I, II, III, and IV are also represented (blue bars in N-terminal domain) [42,63]. The endonuclease active site at the C-terminal end of Pms1 is depicted (blue bar) [39]. The Pms1 interaction region in Mlh1 and the Mlh1 interaction region in the less well-conserved C-terminal domain of Pms1 are also represented (red) [26]. Figure was adapted from [23]. B. Structural alignment of the N-terminal domains of yPms1 and hPMS2. Secondary structure elements are labeled according to that of hPMS2 [22]. Conserved residues between the two proteins are starred and the lid region is underlined in red. Regions with structural differences have tan background. Residues in orange are disordered, residues in pink were mutated in this study, and side chains for the residues colored blue interact with the bound nucleotide. Individual residues that were mutated and are conserved in yMlh1 are underlined in pink.
Figure 2
Figure 2. Ribbon diagrams of the N-terminal domain of yPms1
A. Ribbon diagram of the crystal structure of the N-terminal domain of yPms1, depicted in orange. Amino acids that lie on the surface of the domain are tan and numbered, AMPNPP is magenta and the magnesium ion is green. Secondary structure elements are labeled according to that of hPMS2 [22]. Italicized black numbers represent β-strands, and italicized black letters represent helices. Nt and Ct refer to N- and C- termini. The image was created with Molscript and Raster3D [64,65]. B. Ribbon diagram of the two molecules A (orange) and B (cyan) of the N-terminal domain of yPms1, with bound AMPNPP (green), in the asymmetric unit. The lid domains of A and B molecules are shown in red and blue, respectively. Selected amino acid residues are in black. Nt and Ct refer to N- and C- termini. C. Superposition of molecules A and B reveals differences in the conformation of the lid region. The lid domains of A and B molecules are shown in red and blue, respectively. Selected amino acid residues are represented in black. Ct refers to C-terminus.
Figure 2
Figure 2. Ribbon diagrams of the N-terminal domain of yPms1
A. Ribbon diagram of the crystal structure of the N-terminal domain of yPms1, depicted in orange. Amino acids that lie on the surface of the domain are tan and numbered, AMPNPP is magenta and the magnesium ion is green. Secondary structure elements are labeled according to that of hPMS2 [22]. Italicized black numbers represent β-strands, and italicized black letters represent helices. Nt and Ct refer to N- and C- termini. The image was created with Molscript and Raster3D [64,65]. B. Ribbon diagram of the two molecules A (orange) and B (cyan) of the N-terminal domain of yPms1, with bound AMPNPP (green), in the asymmetric unit. The lid domains of A and B molecules are shown in red and blue, respectively. Selected amino acid residues are in black. Nt and Ct refer to N- and C- termini. C. Superposition of molecules A and B reveals differences in the conformation of the lid region. The lid domains of A and B molecules are shown in red and blue, respectively. Selected amino acid residues are represented in black. Ct refers to C-terminus.
Figure 2
Figure 2. Ribbon diagrams of the N-terminal domain of yPms1
A. Ribbon diagram of the crystal structure of the N-terminal domain of yPms1, depicted in orange. Amino acids that lie on the surface of the domain are tan and numbered, AMPNPP is magenta and the magnesium ion is green. Secondary structure elements are labeled according to that of hPMS2 [22]. Italicized black numbers represent β-strands, and italicized black letters represent helices. Nt and Ct refer to N- and C- termini. The image was created with Molscript and Raster3D [64,65]. B. Ribbon diagram of the two molecules A (orange) and B (cyan) of the N-terminal domain of yPms1, with bound AMPNPP (green), in the asymmetric unit. The lid domains of A and B molecules are shown in red and blue, respectively. Selected amino acid residues are in black. Nt and Ct refer to N- and C- termini. C. Superposition of molecules A and B reveals differences in the conformation of the lid region. The lid domains of A and B molecules are shown in red and blue, respectively. Selected amino acid residues are represented in black. Ct refers to C-terminus.
Figure 3
Figure 3. Ribbon diagrams of the NTDs of yPms1, hPMS2 and E. coli LN40
A. Superposition of molecule A of N-terminal domain yPms1 (orange) onto the structure of hPMS2 (red). Selected amino acid residues are represented in black while italicized black numbers represent β-strands. Ct refers to C-terminus. B. Superposition of molecule A of yPms1 (orange) onto the dimer structure of the LN40 domain of MutL. Figures created with Molscript and Raster3D [64,65]. Positively charged residues are shown in black. Ct refers to C-terminus. C. Superposition of the lid region from molecules A (orange with lid colored red) and B (cyan with lid colored blue) of yPms1 with that of LN40 (grey). Secondary structural elements are labeled as in Figure 1B and Figure 2A. AMPPNP for each molecule is colored according to chain color.
Figure 3
Figure 3. Ribbon diagrams of the NTDs of yPms1, hPMS2 and E. coli LN40
A. Superposition of molecule A of N-terminal domain yPms1 (orange) onto the structure of hPMS2 (red). Selected amino acid residues are represented in black while italicized black numbers represent β-strands. Ct refers to C-terminus. B. Superposition of molecule A of yPms1 (orange) onto the dimer structure of the LN40 domain of MutL. Figures created with Molscript and Raster3D [64,65]. Positively charged residues are shown in black. Ct refers to C-terminus. C. Superposition of the lid region from molecules A (orange with lid colored red) and B (cyan with lid colored blue) of yPms1 with that of LN40 (grey). Secondary structural elements are labeled as in Figure 1B and Figure 2A. AMPPNP for each molecule is colored according to chain color.
Figure 3
Figure 3. Ribbon diagrams of the NTDs of yPms1, hPMS2 and E. coli LN40
A. Superposition of molecule A of N-terminal domain yPms1 (orange) onto the structure of hPMS2 (red). Selected amino acid residues are represented in black while italicized black numbers represent β-strands. Ct refers to C-terminus. B. Superposition of molecule A of yPms1 (orange) onto the dimer structure of the LN40 domain of MutL. Figures created with Molscript and Raster3D [64,65]. Positively charged residues are shown in black. Ct refers to C-terminus. C. Superposition of the lid region from molecules A (orange with lid colored red) and B (cyan with lid colored blue) of yPms1 with that of LN40 (grey). Secondary structural elements are labeled as in Figure 1B and Figure 2A. AMPPNP for each molecule is colored according to chain color.
Figure 4
Figure 4. Electrostatic surface potential of the yPms1 NTD
Electrostatic surface rendering of NTD yPms1 in a similar orientation to that of Figure 3A. The surface potential plot was calculated using the Adaptive Poisson Boltzmann Solver Tool in Pymol [66]. The potential ranges from −8 kTe−1 (red) to 8 kTe−1 (blue). A groove found on the surface is highlighted with a dashed yellow line. Numbers represent positively charged amino acid residues.
Figure 5
Figure 5. Western blot analysis of wild type and mutant Pms1 proteins
The analysis was performed as previously described [54], using extracts of the E134 strain containing the empty pMH8 vector or pMH8 encoding wild type or the designated mutants of yPms1.
Figure 6
Figure 6. DNA binding by the yPms1 NTDs
Proteins examined included wild-type NTD yPms1 and mutant derivatives K197E, R198E, K218E and R243E/K244E. Electrophoretic mobility shift assays were employed to examine binding to A. 41-mer duplex DNA and B. 41-mer ssDNA. These assays used increasing concentrations of yPms1 and a constant substrate DNA concentration (10 nM). % DNA bound was calculated as described in Materials and Methods. In graphs A and B, wild-type NTD yPms1 (open circle), K197E (open square), R198E (open diamond), K218E (dashed line and x) and R243E/K244E (dashed line, closed triangle). C. DNA binding of NTD yPms1 proteins to double-stranded [3H] pGBT9. Proteins examined were wild-type NTD Pms1 and mutants K197E, R198E, K218E and R243E/K244E. Binding assays were performed with a constant protein concentration of 1 µM and excess DNA. Binding reactions were performed in the presence of MgCl2. Protein–DNA binding is presented as percent DNA bound (percentage of total DNA substrate in a given sample that is protein bound). Error bars represent standard errors of the mean for three independent experiments. Error bars are included for all data.
Figure 6
Figure 6. DNA binding by the yPms1 NTDs
Proteins examined included wild-type NTD yPms1 and mutant derivatives K197E, R198E, K218E and R243E/K244E. Electrophoretic mobility shift assays were employed to examine binding to A. 41-mer duplex DNA and B. 41-mer ssDNA. These assays used increasing concentrations of yPms1 and a constant substrate DNA concentration (10 nM). % DNA bound was calculated as described in Materials and Methods. In graphs A and B, wild-type NTD yPms1 (open circle), K197E (open square), R198E (open diamond), K218E (dashed line and x) and R243E/K244E (dashed line, closed triangle). C. DNA binding of NTD yPms1 proteins to double-stranded [3H] pGBT9. Proteins examined were wild-type NTD Pms1 and mutants K197E, R198E, K218E and R243E/K244E. Binding assays were performed with a constant protein concentration of 1 µM and excess DNA. Binding reactions were performed in the presence of MgCl2. Protein–DNA binding is presented as percent DNA bound (percentage of total DNA substrate in a given sample that is protein bound). Error bars represent standard errors of the mean for three independent experiments. Error bars are included for all data.
Figure 6
Figure 6. DNA binding by the yPms1 NTDs
Proteins examined included wild-type NTD yPms1 and mutant derivatives K197E, R198E, K218E and R243E/K244E. Electrophoretic mobility shift assays were employed to examine binding to A. 41-mer duplex DNA and B. 41-mer ssDNA. These assays used increasing concentrations of yPms1 and a constant substrate DNA concentration (10 nM). % DNA bound was calculated as described in Materials and Methods. In graphs A and B, wild-type NTD yPms1 (open circle), K197E (open square), R198E (open diamond), K218E (dashed line and x) and R243E/K244E (dashed line, closed triangle). C. DNA binding of NTD yPms1 proteins to double-stranded [3H] pGBT9. Proteins examined were wild-type NTD Pms1 and mutants K197E, R198E, K218E and R243E/K244E. Binding assays were performed with a constant protein concentration of 1 µM and excess DNA. Binding reactions were performed in the presence of MgCl2. Protein–DNA binding is presented as percent DNA bound (percentage of total DNA substrate in a given sample that is protein bound). Error bars represent standard errors of the mean for three independent experiments. Error bars are included for all data.
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References

    1. Kunkel TA, Erie DA. DNA mismatch repair. Annu Rev Biochem. 2005;74:681–710. - PubMed
    1. Modrich P. Mechanisms in eukaryotic mismatch repair. J Biol Chem. 2006;281:30305–30309. - PMC - PubMed
    1. Jiricny J. The multifaceted mismatch-repair system. Nat Rev Mol Cell Biol. 2006;7:335–346. - PubMed
    1. Hsieh P, Yamane K. DNA mismatch repair: molecular mechanism, cancer, and ageing. Mech Ageing Dev. 2008;129:391–407. - PMC - PubMed
    1. Harfe BD, Jinks-Robertson S. DNA mismatch repair and genetic instability. Annu Rev Genet. 2000;34:359–399. - PubMed

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