DNA Repair Mechanism

Importance

DNA in the living cell is subject to many chemical alterations (a fact often forgotten in the excitement of being able to do DNA sequencing on dried and/or frozen specimens [Link]). If the genetic information encoded in the DNA is to remain uncorrupted, any chemical changes must be corrected.
A failure to repair DNA produces a mutation.
The recent publication of the human genome [Link] has already revealed 130 genes whose products participate in DNA repair. More will probably be identified soon.

Agents that Damage DNA

• Certain wavelengths of radiation
  • ionizing radiation such as gamma rays and X-rays
  • ultraviolet rays, especially the UV-C rays (~260 nm) that are absorbed strongly by DNA but also the longer-wavelength UV-B that penetrates the ozone shield [Link].
• Highly-reactive oxygen radicals produced during normal cellular respiration as well as by other biochemical pathways. [Link to further discussion.]
• Chemicals in the environment
  • many hydrocarbons, including some found in cigarette smoke

    Link to description of a test measuring the mutations caused by the hydrocarbon benzopyrene.

  • some plant and microbial products, e.g. the aflatoxins produced in moldy peanuts
• Chemicals used in chemotherapy, especially chemotherapy of cancers

Types of DNA Damage

1. All four of the bases in DNA (A, T, C, G) can be covalently modified at various positions.
   • One of the most frequent is the loss of an amino group ("deamination") — resulting, for example, in a C being converted to a U.
2. Mismatches of the normal bases because of a failure of proofreading during DNA replication.
   • Common example: incorporation of the pyrimidine U (normally found only in RNA) instead of T.
3. Breaks in the backbone.
   • Can be limited to one of the two strands (a single-stranded break, SSB) or
   • on both strands (a double-stranded break (DSB).
   • Ionizing radiation is a frequent cause, but some chemicals produce breaks as well.
4. Crosslinks Covalent linkages can be formed between bases
   • on the same DNA strand ("intrastrand") or
   • on the opposite strand ("interstrand").
   Several chemotherapeutic drugs used against cancers crosslink DNA [Link].

Repairing Damaged Bases

Damaged or inappropriate bases can be repaired by several mechanisms:

• Direct chemical reversal of the damage
• Excision Repair, in which the damaged base or bases are removed and then replaced with the correct ones in a localized burst of DNA synthesis. There are three modes of excision repair, each of which employs specialized sets of enzymes.
  1. Base Excision Repair (BER)
  2. Nucleotide Excision Repair (NER)
  3. Mismatch Repair (MMR)

The 2015 Nobel Prize in chemistry was shared by three researchers for their pioneering work in DNA repair: Tomas Lindahl (BER), Aziz Sancar (NER), and Paul Modrich (MMR).

Direct Reversal of Base Damage

Perhaps the most frequent cause of point mutations in humans is the spontaneous addition of a methyl group (CH₃-) (an example of alkylation) to Cs followed by deamination to a T. Fortunately, most of these changes are repaired by enzymes, called glycosylases, that remove the mismatched T restoring the correct C. This is done without the need to break the DNA backbone (in contrast to the mechanisms of excision repair described below).
Some of the drugs used in cancer chemotherapy ("chemo") also damage DNA by alkylation. Some of the methyl groups can be removed by a protein encoded by our MGMT gene. However, the protein can only do it once, so the removal of each methyl group requires another molecule of protein.
This illustrates a problem with direct reversal mechanisms of DNA repair: they are quite wasteful. Each of the myriad types of chemical alterations to bases requires its own mechanism to correct. What the cell needs are more general mechanisms capable of correcting all sorts of chemical damage with a limited toolbox. This requirement is met by the mechanisms of excision repair.

Base Excision Repair (BER)

The steps and some key players:

1. removal of the damaged base (estimated to occur some 20,000 times a day in each cell in our body!) by a DNA glycosylase. We have at least 8 genes encoding different DNA glycosylases each enzyme responsible for identifying and removing a specific kind of base damage.
2. removal of its deoxyribose phosphate in the backbone, producing a gap. We have two genes encoding enzymes with this function.
3. replacement with the correct nucleotide. This relies on DNA polymerase beta, one of at least 11 DNA polymerases encoded by our genes.
4. ligation of the break in the strand. Two enzymes are known that can do this; both require ATP to provide the needed energy.

Nucleotide Excision Repair (NER)

NER differs from BER in several ways.

• It uses different enzymes.
• Even though there may be only a single "bad" base to correct, its nucleotide is removed along with many other adjacent nucleotides; that is, NER removes a large "patch" around the damage.

The steps and some key players:

1. The damage is recognized by one or more protein factors that assemble at the location.
2. The DNA is unwound producing a "bubble". The enzyme system that does this is Transcription Factor IIH, TFIIH, (which also functions in normal transcription).
3. Cuts are made on both the 3' side and the 5' side of the damaged area so the tract containing the damage can be removed.
4. A fresh burst of DNA synthesis — using the intact (opposite) strand as a template — fills in the correct nucleotides. The DNA polymerases responsible are designated polymerase delta and epsilon.
5. A DNA ligase covalently inserts the fresh piece into the backbone.

Xeroderma Pigmentosum (XP)

XP is a rare inherited disease of humans which, among other things, predisposes the patient to

• pigmented lesions on areas of the skin exposed to the sun and
• an elevated incidence of skin cancer.

It turns out that XP can be caused by mutations in any one of several genes — all of which have roles to play in NER. Some of them:

• XPA, which encodes a protein that binds the damaged site and helps assemble the other proteins needed for NER.
• XPB and XPD, which are part of TFIIH. Some mutations in XPB and XPD also produce signs of premature aging. [Link]
• XPF, which cuts the backbone on the 5' side of the damage
• XPG, which cuts the backbone on the 3' side.

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