DNA repair mechanisms

DNA repair mechanisms are vital for maintaining genomic stability and preventing mutations that can lead to diseases like cancer. DNA is constantly exposed to damaging agents such as UV radiation, chemicals, and errors during replication, which can cause structural alterations and mispairing of nucleotides. The cell has evolved multiple DNA repair pathways to detect and correct these damages.

1. Types of DNA Damage

DNA damage can be classified into different types based on the nature of the lesion:

• Base Damage: Chemical modifications of DNA bases, such as oxidation, deamination, or alkylation.
• Single-Strand Breaks (SSBs): Breaks in one strand of the DNA double helix.
• Double-Strand Breaks (DSBs): Breaks in both strands of the DNA double helix, which are more severe and can lead to chromosomal fragmentation.
• Mispairing: Incorrect base pairing during DNA replication.
• Crosslinking: Covalent bonds between DNA strands or between DNA and proteins, which can interfere with replication and transcription.

2. Mechanisms of DNA Mispairing

DNA mispairing occurs primarily during replication when an incorrect nucleotide is incorporated into the growing DNA strand. The major types of mispairing include:

• Transition Mismatches: Substitution of a purine for another purine (A ↔ G) or a pyrimidine for another pyrimidine (C ↔ T).
• Transversion Mismatches: Substitution of a purine for a pyrimidine (A ↔ C or G ↔ T).
• Insertion/Deletion Mismatches: Slippage during DNA replication can result in the insertion or deletion of one or more nucleotides.

Mismatch repair (MMR) mechanisms are responsible for correcting these errors after replication.

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3. Major DNA Repair Mechanisms

There are several pathways for DNA repair, each specialized for different types of DNA damage.

3.1. Mismatch Repair (MMR)

Function: Mismatch repair corrects base-pair mismatches and insertion/deletion loops that occur during DNA replication. It ensures the fidelity of DNA replication by identifying and excising mispaired bases.

Steps in Mismatch Repair:

1. Recognition: Mismatch repair proteins, such as MutS in bacteria or MSH2/MSH6 in eukaryotes, recognize the mispaired base.
2. Recruitment of Repair Factors: The MutL complex (or MLH1/PMS2 in eukaryotes) is recruited, along with exonucleases.
3. Excision: The incorrect nucleotide is excised by an exonuclease.
4. Resynthesis: DNA polymerase fills in the gap using the undamaged strand as a template.
5. Ligation: DNA ligase seals the nick to complete the repair.

Significance: Defective mismatch repair is associated with Lynch syndrome, a hereditary cancer predisposition syndrome, and increases the mutation rate in cells.

3.2. Base Excision Repair (BER)

Function: BER corrects small, non-helix-distorting lesions such as oxidized or deaminated bases, alkylated bases, and single-strand breaks. It typically addresses damage caused by reactive oxygen species (ROS), deamination (e.g., cytosine to uracil), and alkylation.

Steps in BER:

1. Damage Recognition: A specific DNA glycosylase enzyme recognizes and removes the damaged base, creating an abasic (AP) site.
2. AP Site Cleavage: AP endonuclease cleaves the DNA backbone at the AP site.
3. Gap Filling: DNA polymerase inserts the correct nucleotide at the site of damage.
4. Ligation: DNA ligase seals the remaining nick.

Types of Lesions Repaired by BER:

• 8-oxoguanine: A common lesion caused by oxidative damage.
• Uracil: Results from the deamination of cytosine.
• Abasic sites: Occur when a base is lost from the sugar-phosphate backbone.

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3.3. Nucleotide Excision Repair (NER)

Function: NER repairs bulky, helix-distorting lesions, such as those caused by UV radiation (e.g., thymine dimers) and chemical adducts (e.g., from smoking or environmental mutagens).

Steps in NER:

1. Damage Recognition: The NER pathway involves two recognition pathways:
   • Global Genomic NER (GG-NER): Recognizes damage throughout the genome.
   • Transcription-Coupled NER (TC-NER): Targets damage that stalls RNA polymerase during transcription.
2. Unwinding: Helicases (such as XPB and XPD in eukaryotes) unwind the DNA around the lesion.
3. Dual Incision: Endonucleases (e.g., XPF and XPG) make incisions on both sides of the lesion to remove a 24–32 nucleotide-long fragment containing the damaged region.
4. Resynthesis: DNA polymerase fills the gap.
5. Ligation: DNA ligase seals the nick.

Significance: Defects in NER are associated with xeroderma pigmentosum (XP), a condition that makes individuals extremely sensitive to UV light and predisposes them to skin cancers.

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3.4. Homologous Recombination (HR)

Function: Homologous recombination repairs double-strand breaks (DSBs) using a homologous sequence as a template. This mechanism is error-free and essential for the maintenance of genomic integrity, particularly during the S and G2 phases of the cell cycle when a sister chromatid is available as a template.

Steps in HR:

1. DSB Detection: The break is recognized by the MRN complex (Mre11, Rad50, Nbs1).
2. End Resection: Nucleases resect the 5’ ends of the broken DNA, leaving 3’ single-stranded overhangs.
3. Strand Invasion: The single-stranded DNA (ssDNA) invades the homologous region of the sister chromatid, facilitated by Rad51, and forms a D-loop.
4. DNA Synthesis: DNA polymerase extends the invading strand using the sister chromatid as a template.
5. Resolution: The Holliday junctions formed during the process are resolved, and the newly synthesized DNA is ligated.

Significance: Defects in homologous recombination, such as mutations in the BRCA1 or BRCA2 genes, increase the risk of breast and ovarian cancers.

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3.5. Non-Homologous End Joining (NHEJ)

Function: NHEJ repairs double-strand breaks without the need for a homologous template, making it an error-prone repair process. It is active throughout the cell cycle but is particularly important during G1.

Steps in NHEJ:

1. DSB Detection: Ku70/Ku80 heterodimer binds to the DNA ends and recruits the DNA-PKcs complex.
2. End Processing: Nucleases trim the DNA ends if necessary, and polymerases fill in missing nucleotides.
3. Ligation: DNA ligase IV, along with co-factors such as XRCC4 and XLF, seals the break.

Significance: NHEJ is more error-prone than HR because it may result in small insertions or deletions at the repair site. However, it is faster and more efficient in non-replicating cells.

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3.6. Direct Reversal Repair

Function: This pathway directly reverses certain types of DNA damage without removing the affected base or nucleotide.

Examples of Direct Reversal:

• O6-methylguanine DNA methyltransferase (MGMT) removes alkyl groups from the O6 position of guanine, which prevents G
  to A
  transitions.
• Photoreactivation: In some organisms (such as bacteria and plants), DNA photolyases use light energy to directly reverse UV-induced pyrimidine dimers.

Significance: Direct reversal repair is a very efficient and error-free repair mechanism but is limited to specific types of DNA damage.

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4. DNA Repair in the Context of Replication and Mispair Mechanisms

Replication errors are a major source of DNA mispairing, where incorrect bases are incorporated during DNA synthesis. The DNA polymerase proofreading function, along with the mismatch repair system, corrects these errors.

Proofreading by DNA Polymerases

• DNA polymerases have 3’ to 5’ exonuclease activity that can remove incorrectly paired nucleotides during replication. If a mispair is detected, the exonuclease activity excises the mispaired base, and the polymerase inserts the correct nucleotide.
• Despite this proofreading function, some errors escape detection, leading to mismatches.

Role of Mismatch Repair in Correcting Replication Errors

Mismatch repair acts as a post-replicative surveillance mechanism. It scans the newly synthesized DNA strand for mismatches that escaped proofreading. Failure in MMR results in an elevated mutation rate and contributes to the development of microsatellite instability (MSI), which is a hallmark of certain types of cancer, particularly colorectal cancer.

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The DNA repair mechanisms in eukaryotic cells are diverse and highly specialized to detect, signal, and repair different types of DNA damage, ensuring genome stability. Mismatch repair is critical for correcting replication errors, while pathways like BER, NER, HR, and NHEJ address various types of DNA lesions. When these repair mechanisms fail or become defective, the resulting genomic instability can lead to cancer and other genetic disorders.