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Related Concept Videos

Mismatch Repair01:36

Mismatch Repair

Overview
Mismatch Repair01:20

Mismatch Repair

Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
The Mutator Protein Family Plays a Key Role in DNA Mismatch Repair
The human genome has more than 3 billion base pairs of DNA per cell. Prior to cell division, that vast amount of genetic...
Mismatch Repair01:36

Mismatch Repair

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Homologous Recombination02:31

Homologous Recombination

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
Overview of DNA Repair02:25

Overview of DNA Repair

In order to be passed through generations, genomic DNA must be undamaged and error-free. However, every day, DNA in a cell undergoes several thousand to a million damaging events by natural causes and external factors. Ionizing radiation such as UV rays, free radicals produced during cellular respiration, and hydrolytic damage from metabolic reactions can alter the structure of DNA. Damages caused include single-base alteration, base dimerization, chain breaks, and cross-linkage.
Chemically...
Overview of DNA Repair02:25

Overview of DNA Repair

In order to be passed through generations, genomic DNA must be undamaged and error-free. However, every day, DNA in a cell undergoes several thousand to a million damaging events by natural causes and external factors. Ionizing radiation such as UV rays, free radicals produced during cellular respiration, and hydrolytic damage from metabolic reactions can alter the structure of DNA. Damages caused include single-base alteration, base dimerization, chain breaks, and cross-linkage.
Chemically...

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Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis
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Eukaryotic DNA mismatch repair in vitro.

Fenghua Yuan1, Limin Song, Fengsong Liu

  • 1Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL, USA. fyuan@med.miami.edu

Methods in Molecular Biology (Clifton, N.J.)
|September 4, 2012
PubMed
Summary

This study details protocols for in vitro mismatch repair assays using yeast and human cell extracts. These methods aid in understanding DNA repair mechanisms and their link to cancer.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • DNA replication generates errors corrected by mismatch repair (MMR).
  • MMR deficiency leads to a mutator phenotype, contributing to hereditary nonpolyposis colorectal cancer and other cancers.
  • High conservation of MMR machinery between yeast and humans allows for cross-species study.

Purpose of the Study:

  • To provide practical protocols for in vitro mismatch repair assays.
  • To enable the study of MMR mechanisms in a cell-free system.
  • To validate a yeast cell-free system for MMR research.

Main Methods:

  • Preparation of yeast and HeLa cell-free nuclear extracts.
  • Generation of site-specific DNA mismatch substrates.
  • In vitro mismatch repair assay using prepared extracts and substrates.

Main Results:

  • Successful establishment of a yeast cell-free system for MMR studies.
  • Validation of the system using a mismatch repair deficient strain (Δmsh2).
  • Complementation assay with purified yeast MutSα confirmed system functionality.

Conclusions:

  • The described protocols facilitate in vitro studies of DNA mismatch repair.
  • The validated yeast cell-free system serves as a valuable tool for investigating MMR mechanisms.
  • This research contributes to understanding DNA repair pathways relevant to human diseases like cancer.