title: A 'Semi-Protected Oligonucleotide Recombination' Assay for DNA Mismatch Repair in vivo Suggests Different Modes of Repair for Lagging Strand Mismatches.
abstract: In Escherichia coli, a DNA mismatch repair (MMR) pathway corrects errors that occur during DNA replication by coordinating the excision and re-synthesis of a long tract of the newly-replicated DNA between an epigenetic signal (a hemi-methylated d(GATC) site or a single-stranded nick) and the replication error after the error is identified by protein MutS. Recent observations suggest that this 'long-patch repair' between these sites is coordinated in the same direction of replication by the replisome. Here, we have developed a new assay that uniquely allows us to introduce targeted 'mismatches' directly into the replication fork via oligonucleotide recombination, examine the directionality of MMR, and quantify the nucleotide-dependence, sequence context-dependence, and strand-dependence of their repair in vivo-something otherwise nearly impossible to achieve. We find that repair of genomic lagging strand mismatches occurs bi-directionally in E. coli and that, while all MutS-recognized mismatches had been thought to be repaired in a consistent manner, the directional bias of repair and the effects of mutations in MutS are dependent on the molecular species of the mismatch. Because oligonucleotide recombination is routinely performed in both prokaryotic and eukaryotic cells, we expect this assay will be broadly applicable for investigating mechanisms of MMR in vivo.
authors.affiliation: Department of Mechanical Engineering and Materials Science, Edmund T. Pratt, Jr. School of Engineering, Duke University, Durham, NC, USA.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Josephs
authors.affiliation: Department of Mechanical Engineering and Materials Science, Edmund T. Pratt, Jr. School of Engineering, Duke University, Durham, NC, USA.
authors.forename: Piotr E
authors.initials: PE
authors.lastname: Marszalek
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D001681:Biological Assay; D004249:DNA Damage; D053843:DNA Mismatch Repair; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D051719:MutS DNA Mismatch-Binding Protein / Q000235:genetics / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism; D011995:Recombination, Genetic
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli