DNA Repair: A Detailed Overview

Why DNA Repair Is Needed: Sources of Damage

"DNA is constantly being subjected to environmental insults that cause the alteration or removal of nucleotide bases... if the damage is not repaired, a permanent change (mutation) is introduced that can result in any of a number of deleterious effects, including loss of control over the proliferation of the mutated cell, leading to cancer." - Biochemistry, 8th ed., Lippincott Illustrated Reviews

Overview: General Repair Steps

1. Recognition - A sensor protein detects the lesion (distorted helix, mismatched base, oxidized base)
2. Excision - An endonuclease nicks the strand; an exonuclease removes the damaged fragment
3. Gap filling - DNA polymerase synthesizes new DNA using the intact complementary strand as template (5'→3' direction)
4. Ligation - DNA ligase seals the remaining nick

Repair Mechanism 1: Direct Repair

• Alkylating agents add methyl or ethyl groups to the O6 position of guanine, causing it to mispair with thymine instead of cytosine
• MGMT transfers the alkyl group from the damaged base directly onto a cysteine in its own active site, restoring normal guanine
• The enzyme is consumed ("suicide enzyme") - one molecule per repair event

• Photolyase enzyme binds the pyrimidine dimer and uses energy from visible light (300-500 nm) to cleave the covalent bonds between the fused pyrimidines
• Restores the individual bases without removing any nucleotides
• Not present in humans

Repair Mechanism 2: Mismatch Repair (MMR)

Steps in E. coli (and analogous process in humans):

• GATC sequences on the parental strand are methylated on adenine by DNA adenine methylase (DAM)
• Immediately after replication, the daughter strand is unmethylated (hemimethylated state)
• MutS protein recognizes the mismatch; MutL is recruited; together they activate MutH, which specifically cleaves the unmethylated (daughter) strand

Repair Mechanism 3: Nucleotide Excision Repair (NER)

Two NER sub-pathways:

Steps in detail (human NER):

1. Damage recognition

• GGR: XPC-RAD23B (with XPE/DDB2 complex) scans the genome and detects helix distortion
• TCR: RNA polymerase stalls at the lesion; CSA and CSB proteins are recruited to signal repair

1. Helix unwinding

• Transcription factor TFIIH is recruited; its helicase subunits XPB (3'→5') and XPD (5'→3') unwind ~25-30 base pairs around the lesion
• XPA stabilizes the open DNA complex and helps verify the damage

1. Dual incision

• XPF-ERCC1 nuclease cuts the damaged strand on the 5' side of the lesion
• XPG nuclease cuts the damaged strand on the 3' side
• A 24-34 nucleotide oligonucleotide containing the lesion is released

1. Gap filling and ligation

• PCNA (sliding clamp), RPA (single-strand binding protein), and DNA polymerase δ/ε fill the gap using the intact complementary strand as template
• DNA ligase seals the final nick

• Xeroderma Pigmentosum (XP): Autosomal recessive; caused by mutations in any of 7 XP genes (XP-A through XP-G). Patients have extreme UV sensitivity and develop skin cancers very early in life.
• Cockayne Syndrome (CS): Mutations in CSA or CSB, affecting transcription-coupled repair specifically. Characterized by premature aging, neurodegeneration, and UV sensitivity without skin cancer.
• Trichothiodystrophy (TTD): Mutations in XPD/XPB; hair, nail, and skin abnormalities.

Repair Mechanism 4: Base Excision Repair (BER)

Steps in detail:

• Specific DNA glycosylases recognize the abnormal base and cleave the N-glycosidic bond between the base and the deoxyribose sugar
• This releases the abnormal base and leaves an abasic (AP) site - an apurinic or apyrimidinic site
• Examples: Uracil-DNA glycosylase removes uracil (from C deamination); OGG1 removes 8-oxoguanine (oxidative damage); MYH glycosylase removes adenine misincorporated opposite 8-oxoG

• AP endonuclease (APE1) cleaves the phosphodiester backbone 5' to the AP site, creating a nick with a 3'-OH and a 5'-deoxyribose phosphate (dRP)
• DNA polymerase β (in short-patch BER) removes the dRP group using its lyase activity and inserts one correct nucleotide

• Short-patch BER (most common): Pol β inserts a single nucleotide; XRCC1-DNA ligase III seals the nick
• Long-patch BER: Pol δ/ε displaces 2-10 nucleotides (strand displacement synthesis); the displaced "flap" is cleaved by FEN1; DNA ligase I seals the nick

• Mutations in MYH (the DNA glycosylase that removes adenine mispaired with 8-oxoguanine) cause MAP - MUTYH-associated polyposis, an autosomal recessive form of multiple colorectal adenomas/cancer

Repair Mechanism 5: Double-Strand Break (DSB) Repair

5a. Homologous Recombination (HR)

• Requires a homologous DNA template (sister chromatid or homologous chromosome)
• Therefore only active in S and G2 phases of the cell cycle (after replication)
• High fidelity - restores the exact sequence

1. End resection: The MRN complex (MRE11-RAD50-NBS1) and CtIP resect the broken ends to produce long 3' single-stranded overhangs
2. RPA coating: RPA (replication protein A) coats the single-stranded DNA to protect it
3. RAD51 loading: BRCA2 facilitates loading of RAD51 recombinase onto the ssDNA, displacing RPA. BRCA1 helps recruit the repair machinery.
4. Strand invasion: The RAD51-coated single strand invades the homologous duplex (usually the sister chromatid), forming a D-loop (displacement loop)
5. DNA synthesis: DNA polymerase extends from the 3'-OH end using the sister chromatid as template
6. Resolution: The Holliday junction intermediates are resolved by resolvases/dissolverases, restoring two intact duplexes

5b. Non-Homologous End Joining (NHEJ)

• Does not require a homologous template
• Active in all phases of the cell cycle (especially G1)
• Error-prone - can cause small insertions or deletions (indels) at the junction
• The predominant DSB repair pathway in mammals

1. End recognition: Ku70/Ku80 heterodimer rapidly binds both broken DNA ends and protects them from degradation
2. Recruitment of DNA-PKcs: The Ku complex recruits DNA-dependent protein kinase catalytic subunit (DNA-PKcs), forming the DNA-PK holoenzyme. DNA-PK synapses the two ends together.
3. End processing: Nucleases (Artemis, activated by DNA-PKcs) and polymerases (pol μ, pol λ) trim and fill in the ends as needed to make them compatible for joining
4. Ligation: The XRCC4-DNA Ligase IV complex (with XLF/Cernunnos) ligates the two ends together

Repair Mechanism 6: Translesion Synthesis (TLS) - Damage Tolerance

• Normal replicative polymerases stall at bulky lesions
• TLS polymerases (pol η, pol ι, pol κ, REV1) have larger, more flexible active sites that can accommodate distorted templates
• They insert nucleotides across the lesion, often with reduced fidelity (mutagenic bypass)
• Pol η specifically inserts AA opposite thymine dimers - this is essentially error-free bypass
• Xeroderma Pigmentosum Variant (XP-V) is caused by mutations in pol η - patients can repair dimers via NER but cannot perform error-free bypass, leading to increased UV mutagenesis

Integration with the Cell Cycle: The DNA Damage Response (DDR)

1. DNA damage is sensed by the MRN complex (DSBs) or RPA-loaded ssDNA (replication stress)
2. ATM kinase (activated by DSBs) or ATR kinase (activated by ssDNA/stalled replication forks) are recruited
3. ATM/ATR phosphorylate and activate checkpoint kinases CHK2 and CHK1, respectively
4. These kinases phosphorylate CDC25 phosphatases, inactivating them, which prevents activation of cyclin-CDK complexes - cell cycle arrest
5. ATM/ATR also activate p53 (TP53), which transcriptionally induces p21 (a CDK inhibitor) for sustained arrest, and BAX/PUMA for apoptosis if damage is irreparable
6. Repair then proceeds; once complete, the checkpoint is lifted and the cell cycle resumes

Summary Table

• Biochemistry, 8th ed., Lippincott Illustrated Reviews, Ch. 30 (DNA repair, pp. 1176-1187)
• Basic Medical Biochemistry: A Clinical Approach, 6e, Ch. 12 (DNA damage & repair, pp. 411-417)
• Emery's Elements of Medical Genetics and Genomics, p. 34 (Table 2.7 - DNA repair pathways)
• Henry's Clinical Diagnosis and Management by Laboratory Methods, Ch. 68 (Table 68.3 - DNA Repair Pathways)
• Campbell-Walsh-Wein Urology, Ch. 33 (DNA Damage Response)
• Fitzpatrick's Dermatology, Ch. 17 (UV-induced DNA damage and NER, pp. 307-310)