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A 'Semi-Protected Oligonucleotide Recombination' Assay for DNA Mismatch Repair in vivo Suggests Different Modes of Repair for Lagging Strand Mismatches.

Publication ,  Journal Article
Josephs, EA; Marszalek, PE
Published in: Nucleic acids research
May 2017

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.

Duke Scholars

Published In

Nucleic acids research

DOI

EISSN

1362-4962

ISSN

0305-1048

Publication Date

May 2017

Volume

45

Issue

8

Start / End Page

e63

Related Subject Headings

  • Recombination, Genetic
  • Oligonucleotides
  • MutS DNA Mismatch-Binding Protein
  • Gene Expression Regulation, Bacterial
  • Escherichia coli Proteins
  • Escherichia coli
  • Developmental Biology
  • DNA, Bacterial
  • DNA Replication
  • DNA Mismatch Repair
 

Citation

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Josephs, E. A., & Marszalek, P. E. (2017). A 'Semi-Protected Oligonucleotide Recombination' Assay for DNA Mismatch Repair in vivo Suggests Different Modes of Repair for Lagging Strand Mismatches. Nucleic Acids Research, 45(8), e63. https://doi.org/10.1093/nar/gkw1339
Josephs, Eric A., and Piotr E. Marszalek. “A 'Semi-Protected Oligonucleotide Recombination' Assay for DNA Mismatch Repair in vivo Suggests Different Modes of Repair for Lagging Strand Mismatches.Nucleic Acids Research 45, no. 8 (May 2017): e63. https://doi.org/10.1093/nar/gkw1339.
Josephs, Eric A., and Piotr E. Marszalek. “A 'Semi-Protected Oligonucleotide Recombination' Assay for DNA Mismatch Repair in vivo Suggests Different Modes of Repair for Lagging Strand Mismatches.Nucleic Acids Research, vol. 45, no. 8, May 2017, p. e63. Epmc, doi:10.1093/nar/gkw1339.
Journal cover image

Published In

Nucleic acids research

DOI

EISSN

1362-4962

ISSN

0305-1048

Publication Date

May 2017

Volume

45

Issue

8

Start / End Page

e63

Related Subject Headings

  • Recombination, Genetic
  • Oligonucleotides
  • MutS DNA Mismatch-Binding Protein
  • Gene Expression Regulation, Bacterial
  • Escherichia coli Proteins
  • Escherichia coli
  • Developmental Biology
  • DNA, Bacterial
  • DNA Replication
  • DNA Mismatch Repair