DNA Modification
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  • DNA Repair

    DNA is susceptible to degradation from a number of sources, including oxidation, UV radiation, ionizing radiation, phenol/chloroform extraction, mechanical shearing, formalin fixation (post extraction), or long term storage. DNA repair proteins and processes can facilitate the investigative analysis of environmental samples, ancient DNA, DNA damage control, and DNA-DNA and protein-DNA interactions. DNA repair can also be used to prepare DNA for downstream applications such as PCR, microarray analysis, or other DNA technologies.

    There are several DNA repair mechanisms that a cell utilizes to constantly check for, and repair, damaged DNA. Repair mechanisms are generally specific to a class of DNA damage.

    • Base-excision repair removes bases that have been damaged by oxidation, ionizing radiation, or deamination. Damaged bases are recognized and excised by a DNA glycosylase by cleaving the bond of the base to the deoxyribose, leaving the apyrimidinic or apurinic (AP) site – a sugar not bonded to a base. These sites are repaired by an AP endonuclease, which cleaves phosphodiester bond adjacent to the AP site. DNA glycosylases-lyases typically couple the glycosylase activity and phosphodiester bond breaking activity, referred to as lyase activity. Some DNA glycosylases-lyases can excise the damaged base and subsequently remove the base-free deoxyribose moiety thus leaving DNA with a single nucleotide gap.
    • Nucleotide-excision repair is a mechanism by which deformation of the delicate DNA structure, caused by UV-induced pyrimidine dimers or the addition of bulky functional groups, is recognized and excised as part of an oligonucleotide. Damage is recognized by a complex of proteins and unwound by helicases. Endonucleases cleave an unwound DNA strand containing the damage.
    • Mismatch repair recognizes mismatched bases incorporated during DNA replication. Though the proofreading activity of DNA polymerase removes most of the incorrectly incorporated bases during replication, mismatch repair is a redundant system that scans the newly synthesized daughter DNA and excises the mismatched base.

    In each repair mechanism, the gap formed by excision of the nucleotides(s) is filled by DNA polymerase and sealed by DNA ligase. (1)

    There are a number of different techniques used to identify DNA damage:

    • Single cell gel electrophoresis assay (also known as comet assay) encases cells in a low-melting-point agarose suspension, where they are lysed in alkaline conditions, and electrophoresed on a slide. The DNA is made visible by the use of a fluorescent stain and a visual analysis is used to determine the extent of DNA damage. (2)
    • Alkaline elution provides a sensitive measure of DNA single-strand breakage by measuring the rate of elution of single-stranded DNA from lysed cells on membrane filters. The rate of elution is proportional to the length of DNA and the extent of damage. (3)
    • Alkaline unwinding is the use of an alkaline environment to unwind DNA at single-stranded break sites. The sample is passed through a hydroxyapatite column and the eluent is incubated with a fluorescent marker. The fluorescence reading indicates the number of DNA breaks by quantifying the amount of unwound single-stranded DNA present. (4)

    References

    1. Cooper, Geoffery M. The Cell: A Molecular Approach. 4th ed. Washington D.C.: ASM Press, 2007. 216-223.
    2. Collins, A. et al. (1996) Environmental Health Perspectives, 104, 465-469. PMID: 8781365
    3.  Pflaum, M. et al. (1998) Free Rad. Res., 29, 585-594. PMID: 10098463
    4. Hartwig, A. et al. (1996) Toxicology Letters, 88, 85-90. PMID: 8920721