MutS interaction with mismatch and alkylated base containing DNA molecules detected by optical biosensor.
An optical biosensor was used to monitor interactions between the Escherichia coli DNA mismatch repair molecule MutS and various immobilized oligonucleotides. While associating poorly with single-stranded DNA, MutS was capable of rapid association/dissociation from homoduplex DNA. The interaction of MutS with oligonucleotide 30-mers containing single site mismatches demonstrated that during the dissociation phase, MutS binding was greatest to a G-G mismatch, followed by G-T > A-A > C-T, A-C. Binding to A-G, T-T and C-C mispairs was marginally higher than that seen between MutS and homoduplex DNA. The ability of MutS to interact with 30-mers containing alkylated bases was also tested. While binding to O6-methyl-G-C, or to O4-methyl-T-A base pairs was similar to that of homoduplex DNA, strong binding was seen to a O6-methyl-G-T mispair. O4-methyl-T-G, however, was poorly recognized by MutS, with relative binding affinity similar to homoduplex DNA, predicting poor in vivo recognition of O4-methyl-T-G by MutS. Interestingly, MutS demonstrated a relatively high affinity for an 1,N6-etheno-A-T containing homoduplex. Thus, in allowing rapid evaluation of interactions between such molecules, the biosensor will be useful to structure-function analyses.
Babic
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Biomedical Research Center, University of British Columbia, Vancouver, Canada.
Andrew
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Jirik
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D000251:Adenosine Triphosphatases; D000478:Alkylation; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001482:Base Composition; D015374:Biosensing Techniques; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins; D003838:Deoxyadenine Nucleotides / Q000737:chemistry / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins; D051719:MutS DNA Mismatch-Binding Protein; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism*; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D003838:Deoxyadenine Nucleotides; D003848:Deoxyguanine Nucleotides; D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli