title: Modeling of the DNA-binding site of yeast Pms1 by mass spectrometry.
abstract: Mismatch repair (MMR) corrects replication errors that would otherwise lead to mutations and, potentially, various forms of cancer. Among several proteins required for eukaryotic MMR, MutLα is a heterodimer comprised of Mlh1 and Pms1. The two proteins dimerize along their C-terminal domains (CTDs), and the CTD of Pms1 houses a latent endonuclease that is required for MMR. The highly conserved N-terminal domains (NTDs) independently bind DNA and possess ATPase active sites. Here we use two protein footprinting techniques, limited proteolysis and oxidative surface mapping, coupled with mass spectrometry to identify amino acids involved along the DNA-binding surface of the Pms1-NTD. Limited proteolysis experiments elucidated several basic residues that were protected in the presence of DNA, while oxidative surface mapping revealed one residue that is uniquely protected from oxidation. Furthermore, additional amino acids distributed throughout the Pms1-NTD were protected from oxidation either in the presence of a non-hydrolyzable analog of ATP or DNA, indicating that each ligand stabilizes the protein in a similar conformation. Based on the recently published X-ray crystal structure of yeast Pms1-NTD, a model of the Pms1-NTD/DNA complex was generated using the mass spectrometric data as constraints. The proposed model defines the DNA-binding interface along a positively charged groove of the Pms1-NTD and complements prior mutagenesis studies of Escherichia coli and eukaryotic MutL.
authors.affiliation: Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, United States. schorzmana@niehs.nih.gov
authors.forename: Allison N
authors.initials: AN
authors.lastname: Schorzman
authors.forename: Lalith
authors.initials: L
authors.lastname: Perera
authors.forename: Jenny M
authors.initials: JM
authors.lastname: Cutalo-Patterson
authors.forename: Lars C
authors.initials: LC
authors.lastname: Pedersen
authors.forename: Lee G
authors.initials: LG
authors.lastname: Pedersen
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Kunkel
authors.forename: Kenneth B
authors.initials: KB
authors.lastname: Tomer
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D053843:DNA Mismatch Repair / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015003:Yeasts / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D010455:Peptides; D004247:DNA