DNA Repair Mechanisms

Although DNA fidelity is highly protected, DNA can still be damaged by a number of environmental factors, reactive oxygen species, and errors in DNA replication. DNA repair is a continuous process in which the cell corrects the damage. The cell has multiple mechanisms it can use to repair DNA. During replication, the cell has proofreading machinery within the DNA polymerase itself. For single-stranded DNA damage, the cell can use excision repair techniques and photorepair. For double-stranded DNA breaks, the cell can use homologous recombination or nonhomologous end joining. When normal DNA repair processes fail owing to age, dysfunction, or an overloaded system, unrepaired DNA damage can lead to apoptosis, cellular senescence, or malignant tumors.

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Overview of DNA Damage and Repair

Etiology

DNA damage can be caused by:

  • Environmental exposures:
    • Sunlight/UV radiation
    • Radiation
    • Chemicals
    • Viruses
    • Diet
  • Reactive oxygen species (ROS) produced during normal metabolic processes
  • Replication errors
  • Age

Types of DNA damage

  • Single-stranded damage: 
    • Damage to 1 strand allows use of the other strand as a template for repair.
    • Examples of single-stranded damage include:
      • Breaks in 1 strand of the DNA
      • Mismatched base pairs
      • Incorporation of uracil into the DNA
      • Oxidation or alkylation of the DNA 
  • Double-stranded breaks:
    • Both strands of DNA are severed.
    • Dangerous to genome stability (chromosomal damage)
    • Cross-linking of broken strands can lead to arrest of mitosis, cell death, or mutation.

DNA repair mechanisms

  • Mechanisms used to correct replication errors during DNA replication: 
    • Proofreading
  • Mechanisms for single-stranded DNA damage (after replication):
    • Photorepair (not active in humans)
    • Base excision repair
    • Nucleotide excision repair
    • Mismatch repair
  • Mechanisms for double-stranded DNA breaks (after replication):
    • Homologous recombination
    • Nonhomologous end joining

Proofreading and Photorepair

Proofreading

Proofreading occurs during DNA replication. DNA polymerase (the enzyme complex that replicates DNA):

  • Has “proofreading” activity, capable of correcting replication errors
  • Can “back up” when an error is detected, excise the incorrect nucleotide, and insert the correct one:
    • Has 3’ to 5’ exonuclease activity allowing this excision of the incorrect nucleotide
  • Improves the fidelity of copying the DNA 100-fold
Proofreading activity of dna polymerase

Proofreading activity of DNA polymerase

Image by Lecturio.

Photorepair

UV light radiation causes pyrimidine (T or C) dimers to form via covalent linkages between adjacent bases, creating a conformational change (“bulge”) in the DNA. These defects can be repaired via a process called photorepair, or photoreactivation.

  • DNA photolyase is an enzyme that uses visible light energy to break the covalent bond between pyrimidines, restoring the DNA to its original state:
    • Note that UV light causes the damage; visible light provides the energy to fix the damage.
  • Photorepair is an example of direct (or chemical) reversal of DNA damage (i.e., it does not break the phosphodiester backbone).
  • Does not need a DNA template
  • Can repair only 1 base at a time
  • This process is not active in placental mammals (such as humans).
Steps of dna photorepair

Steps of photorepair

Image by Lecturio.

Single-Stranded DNA Damage Repair

The cell has 3 primary mechanisms to repair damage to a single strand of DNA: base excision repair, nucleotide excision repair, and mismatch repair.

General process of single-stranged DNA damage repair

All 3 mechanisms follow the same general process:

  1. The damaged or abnormally paired bases are identified.
  2. Excision enzymes excise the abnormal base, nucleotide, or a small region surrounding it.
  3. DNA polymerase fills in the gap by adding the correct nucleotides.
  4. DNA ligase seals the sugar-phosphate backbone with a new phosphodiester bond.
General mechanism of single-stranded dna repair

General mechanism of single-stranded DNA repair

Image by Lecturio.

Base excision repair (BER)

In BER, a single damaged base is excised and replaced.

  • Glycosylase enzymes:
    • Cleave the bond between the damaged base and deoxyribose, removing it
    • DNA glycosylases are lesion-specific.
    • Cells have multiple DNA glycosylases with different specificities.
    • Examples of glycosylase enzymes:
      • Uracil-DNA glycosylase removes uracil from DNA strands.
      • Oxoguanine glycosylase removes oxidized bases.
  • Endonucleases remove the remaining sugar-phosphate portion of the nucleotide.
  • DNA polymerase removes the single abnormal nucleotide and replaces it with the correct one.
  • DNA ligase seals the backbone.
  • Mechanism of UV damage repair in humans

Nucelotide excision repair (NER)

  • Similar process to BER, but with larger sections of DNA excised
  • The entire section of DNA (typically, approximately 12–30 base pairs) surrounding the abnormal area is removed by a complex of endonucleases.
  • DNA is resynthesized by DNA polymerase and sealed by DNA ligase.

DNA mismatch repair (MMR)

  • Corrects errors in base pairing that occurred during DNA replication and were not repaired by proofreading:
    • Often caused by tautomerization (structural isomers) during replication
    • Can be single base pairs or larger sections of DNA
  • MMR proteins:
    • Locate the mismatched nucleotides
    • Recruit exonucleases to excise the mismatched bases
  • Once the mismatched base is excised:
    • DNA polymerase can place the correct base(s).
    • DNA ligase seals the sugar-phosphate backbone.

Double-Stranded DNA Damage Repair

In general, double-stranded DNA damage is harder to repair because there is no template strand to work off of. The 2 primary mechanisms to fix double-stranded DNA breaks are homologous recombination and nonhomologous end joining.

Homologous recombination (HR)

In homologous recombination (HR), the nearly identical sister chromatid or homologous chromosome is used as a template:

  • Mechanics and enzymes are similar to the chromosomal crossing over that occurs during meiosis.
  • HR requires:
    • Extensive regions of homologous sequences between the 2 chromatids
    • Multiple enzyme complexes, including:
      • Exonucleases: begin digesting the 5’ ends → generates 3’ single-stranded DNA tails on the broken strands
      • Recombinase enzymes: catalyze the insertion of the 3’ end into the complementary sequence of DNA on the homologous chromatid
  • DNA polymerase is able to synthesize new DNA from the homologous chromatid.
  • The “invading” strand may end up:
    • Being exchanged with its homologous region on the opposite chromatid: 
      • This exchange is known as a Holliday junction, a second end capture, or crossing over.
    • Being displaced after it has synthesized enough DNA to “cross the gap” of its original break:
      • The original 3’ end is then reconnected with its original 5’ end and used as a template to fill in the gaps.
      • Known as synthesis-dependent single-strand annealing (SDSA)
    • Copying the entire rest of its sister chromatid if its original 5’ end is lost:
      • Once the invading 3’ end has copied the rest of the chromatid, it releases and is used as a template strand to remake its complementary strand.
      • This process is known as break-induced replication (BIR).
Models of homologous recombination

Models of homologous recombination:
Double-stranded breaks can be repaired using the homologous recombination machinery in a variety of ways. The DNA ends are first processed into 3′ single-stranded DNA tails. These tails invade a homologous template (red), priming new DNA synthesis (dashed line). Shown are 3 possible outcomes from this invasion.
A: In canonical double-stranded break repair (DSBR), both the initial invading strand and the captured second end anneal to the homologous template and prime new DNA synthesis, resulting in a double Holliday junction that can be resolved by nucleases into a crossover or a noncrossover product (noncrossover product shown).
B: Alternatively, after the single-stranded DNA tail invades the homologous template, a round of DNA synthesis is prepared from the 3′ end (dashed red line). Synthesis-dependent strand annealing (SDSA) occurs when the invading strand, along with the newly synthesized segment, is unwound by a helicase and annealed with the other resected end.
C: In break-induced replication (BIR), 1 end of the DSB is lost and the remaining end invades the homologous template priming DNA synthesis to the end of the chromosome.

Image: “Models of homologous recombination” by Jacqueline H. Barlow and Rodney Rothstein. License: CC BY 2.0

Nonhomologous end joining (NHEJ)

  • DNA ligase IV and a cofactor, XRCC4, directly join the broken ends back together.
  • Relies on microhomologies (short homologous sequences) on single-stranded tails of the broken strands
  • Deletions, insertions, and translocations are more common with this type of repair.
  • A similar mechanism is involved in V(D)J recombination to create B-cell and T-cell diversity.

Clinical Relevance

  • Genetic disorders: When mismatched base pairs are not repaired within the gametes, they can create mutations that are passed on to offspring.
  • Cancer: class of diseases caused by changes in the genetic sequence and gene expression. These changes are often due to DNA mutations or damage. There are 4 classes of normal regulatory genes that are often damaged in cancer cells: growth-promoting oncogenes, growth-inhibiting tumor suppressor genes, genes that regulate apoptosis, and genes involved in DNA repair.
  • Lynch syndrome: also called hereditary nonpolyposis colorectal cancer (HNPCC). Lynch syndrome is caused by an autosomal dominant mutation in DNA MMR genes. Lynch syndrome is the most common inherited colon cancer syndrome, and it carries a significantly increased risk for endometrial cancer and other malignancies. Diagnosis is made by genetic testing. Early and frequent screening for colon cancer is required, and prophylactic hysterectomy is often recommended for women beyond reproductive age. 
  • Skin cancers: The most common types of skin cancers include basal cell carcinoma, squamous cell carcinoma of the skin, and melanoma. The mutations in these cancers often arise from DNA damage due to excessive exposure to UV radiation. When overloading of BER systems occurs owing to frequent sun exposure, the risk for skin cancers increase. This risk is most common in fair-skinned individuals with a history of excessive sun exposure and sunburns. Definitive diagnosis is established with biopsy. Treatment relies primarily on surgical excision.
  • Xeroderma pigmentosum: rare autosomal recessive disorder characterized by pigment changes in the skin. The presence of xeroderma pigmentosum leads to an increased risk for UV-induced skin and mucous membrane cancers and, occasionally, progressive neurodegeneration. This disorder is caused by 1 of several mutations, which ultimately leads to dysfunctional NER. Diagnosis should be suspected in children with severe sun sensitivity, significant freckling prior to age 2, and/or skin cancer prior to age 10.

References

  1. Friedberg, E. (2003). DNA damage and repair. Nature 421:436–440. https://doi.org/10.1038/nature01408
  2. Li, X., Heyer, W. D. (2008). Homologous recombination in DNA repair and DNA damage tolerance. Cell Research 18:99–113. https://doi.org/10.1038/cr.2008.1
  3. Roldan-Arjona, T., Ariza, R. R., Cordoba-Canero, D. (2019). DNA base excision repair in plants: an unfolding story with familiar and novel characters. Frontiers in Plant Science. Retrieved April 23, 2021, from https://www.frontiersin.org/articles/10.3389/fpls.2019.01055/full

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