13.3 DNA damage and repair mechanisms

Types of DNA Damage and Repair Mechanisms

Every cell in your body accumulates tens of thousands of DNA lesions per day. Environmental exposures like UV light and endogenous processes like normal metabolism constantly damage your genetic material. Cells survive because they have multiple, overlapping repair systems, each specialized for a different kind of damage. When those systems fail, the result is mutations, genomic instability, and disease.

Types of DNA Damage

Base modifications are the most common form of damage. Chemical reactions alter individual bases without breaking the DNA backbone. Three major types:

• Oxidation adds oxygen groups to bases. The most frequent product is 8-oxoguanine, generated by reactive oxygen species (ROS) from normal aerobic metabolism.
• Deamination removes an amino group. Cytosine spontaneously deaminates to uracil roughly 100–500 times per cell per day. If left unrepaired, the next round of replication pairs uracil with adenine instead of guanine, creating a C→T point mutation.
• Alkylation adds methyl or other alkyl groups to bases, which can block replication or cause mispairing.

Single-strand breaks (SSBs) sever one strand of the double helix. ROS and ionizing radiation are common causes. Because the complementary strand remains intact, SSBs are generally easier to repair.

Double-strand breaks (DSBs) sever both strands and are the most dangerous lesion. Causes include ionizing radiation, replication fork collapse, and certain chemotherapeutic agents (e.g., cisplatin, etoposide). A single unrepaired DSB can trigger cell death or major chromosomal rearrangements.

DNA Repair Mechanisms

Base Excision Repair (BER)

BER fixes small, non-helix-distorting base modifications like oxidized or deaminated bases. It's the most frequently used repair pathway.

1. A DNA glycosylase specific to the type of damage recognizes and cleaves the damaged base, leaving the sugar-phosphate backbone intact. This creates an apurinic/apyrimidinic (AP) site, a position with no base.
2. AP endonuclease cuts the backbone at the AP site, generating a single-strand nick.
3. DNA polymerase β (in short-patch BER) fills in the correct nucleotide using the undamaged strand as a template.
4. DNA ligase III seals the remaining nick, restoring an intact strand.

The key concept: BER removes just the damaged base first, then fixes the backbone. It's a minimal, precise repair.

Nucleotide Excision Repair (NER)

NER handles bulky, helix-distorting lesions that BER cannot, such as thymine dimers caused by UV light or large chemical adducts. Instead of removing a single base, NER excises an entire short stretch of nucleotides (typically 24–32 nucleotides in eukaryotes).

1. Damage recognition occurs through one of two sub-pathways:

   • Global genome NER (GG-NER) scans the entire genome for distortions.
   • Transcription-coupled NER (TC-NER) is triggered when RNA polymerase stalls at a lesion during transcription, giving actively transcribed genes priority repair.

2. Dual incisions are made on either side of the lesion, and the damaged oligonucleotide is removed.

3. DNA polymerase (Pol δ or Pol ε) fills the gap using the undamaged strand as a template.

4. DNA ligase I seals the nick.

Defects in NER cause xeroderma pigmentosum (XP), a disorder in which patients are extremely sensitive to UV light and have a dramatically increased risk of skin cancer.

Mismatch Repair (MMR)

MMR corrects replication errors that escape proofreading by DNA polymerase, including base-base mismatches and small insertion/deletion loops (IDLs). It improves replication fidelity by roughly 100- to 1000-fold.

1. MutS homologs (MSH2/MSH6 for mismatches; MSH2/MSH3 for IDLs) recognize the error.
2. MutL homologs (primarily MLH1/PMS2 in humans) are recruited and help identify the newly synthesized strand. In E. coli, strand discrimination relies on MutH recognizing unmethylated GATC sites on the new strand; in eukaryotes, strand nicks associated with Okazaki fragments and the 3' end of the leading strand serve as discrimination signals.
3. An exonuclease (EXO1) degrades the error-containing strand back past the mismatch.
4. DNA polymerase δ resynthesizes the correct sequence.
5. DNA ligase I seals the nick.

Loss of MMR (particularly MLH1 or MSH2) causes Lynch syndrome (hereditary nonpolyposis colorectal cancer) and produces a hallmark molecular signature called microsatellite instability (MSI), where short repetitive sequences expand or contract due to uncorrected slippage errors.

DNA Damage Response and Consequences

DNA Damage Checkpoints

When damage is detected, cells activate checkpoint pathways that halt the cell cycle and buy time for repair. Two master kinases sit at the top of these signaling cascades: ATM (responds primarily to DSBs) and ATR (responds primarily to stretches of single-stranded DNA, such as stalled replication forks).

G1/S checkpoint prevents cells with damaged DNA from entering S phase and replicating a damaged template.

• DNA damage activates ATM/ATR, which stabilize and activate the p53 tumor suppressor.
• p53 induces transcription of p21, a cyclin-dependent kinase inhibitor.
• p21 inhibits Cyclin E/CDK2, blocking the G1→S transition.

Intra-S checkpoint slows replication origin firing when damage is encountered during S phase.

• ATR senses RPA-coated single-stranded DNA at stalled forks and activates Chk1 kinase.
• Chk1 phosphorylates and degrades Cdc25A, preventing activation of CDK2 and slowing new origin firing.

G2/M checkpoint prevents cells from entering mitosis with unrepaired damage.

• ATM activates Chk2 kinase in response to DSBs.
• Chk2 phosphorylates Cdc25C, causing its cytoplasmic sequestration. Without Cdc25C in the nucleus, Cyclin B/CDK1 remains inhibited and mitotic entry is blocked.

If damage is too severe to repair, these same pathways can push the cell toward apoptosis (through p53-dependent induction of pro-apoptotic genes like BAX and PUMA) or senescence (permanent cell cycle arrest). Both outcomes prevent a damaged cell from proliferating.

Consequences of Unrepaired Damage

When repair mechanisms fail or are overwhelmed, unrepaired lesions have three major consequences:

Mutations are permanent changes to the DNA sequence.

• Point mutations include base substitutions (transitions and transversions), small insertions, and small deletions. These can alter protein function, create premature stop codons, or disrupt regulatory elements.
• Chromosomal aberrations are larger-scale changes: translocations, large deletions, duplications, and inversions. These often result from misrepaired DSBs.

Genomic instability is an increased rate of acquiring new mutations and chromosomal changes. Two common forms:

• Microsatellite instability (MSI) results from defective mismatch repair. Repetitive sequences accumulate insertion/deletion errors, and MSI is a diagnostic marker for Lynch syndrome.
• Chromosomal instability (CIN) involves frequent gains, losses, or rearrangements of whole chromosomes or large segments. CIN is a hallmark of many solid tumors.

Disease outcomes of persistent genomic instability include:

• Cancer, driven by the accumulation of mutations in oncogenes (gain-of-function) and tumor suppressor genes (loss-of-function). Most cancers require multiple "hits" across these genes.
• Premature aging syndromes (progeroid syndromes) such as Werner syndrome (defective WRN helicase) and Cockayne syndrome (defective TC-NER), where impaired DNA repair accelerates tissue deterioration.
• Inherited genetic disorders when germline mutations in repair genes are passed to offspring. Examples include Fanconi anemia (defective interstrand crosslink repair) and Bloom syndrome (defective BLM helicase, leading to elevated sister chromatid exchange).