Premed · Premed · Genetics
Lecture 9: DNA Repair Pathways
Genetics
Learning Objectives
By the end of this lecture, students will be able to:
- Explain why DNA repair is essential for genome integrity and cell survival
- Describe the mechanisms of direct reversal repair, base excision repair, nucleotide excision repair, and mismatch repair
- Explain the repair of double-strand breaks by homologous recombination and non-homologous end joining
- Describe translesion synthesis and its role as a damage tolerance mechanism
- Connect defective DNA repair pathways to human disease (xeroderma pigmentosum, Lynch syndrome, BRCA-related cancers)
- Explain the SOS response in bacteria
Lecture Content
I. Overview of DNA Damage and Repair
DNA is constantly under assault from both endogenous and exogenous sources, with an estimated 10,000 to 100,000 lesions occurring per cell per day. Without repair, mutations accumulate, leading to cancer, cell death, and aging. To combat this, cells have evolved multiple repair pathways, each specialized for different types of damage.
The general repair strategy follows a common logic: first the damage is recognized, then it is removed or reversed, the resulting gap is filled using the complementary strand as a template, and finally the backbone is sealed by ligation. When damage is too extensive for repair, cells can either tolerate it through bypass mechanisms or trigger apoptosis to prevent the propagation of dangerous mutations.
II. Direct Reversal Repair
Direct reversal is the simplest repair mechanism, in which damage is chemically reversed without excising bases. Photoreactivation uses the enzyme photolyase, which harnesses visible light energy to directly cleave pyrimidine dimers. This highly efficient pathway is present in bacteria, plants, and many animals but notably absent in placental mammals, including humans.
O6-methylguanine-DNA methyltransferase (MGMT, also known as Ada protein in bacteria) removes methyl or ethyl groups from the O6 position of guanine by transferring the alkyl group to a cysteine residue on the protein itself. This makes MGMT a "suicide enzyme," as each molecule can act only once before being degraded. MGMT is important for preventing G:C to A:T transitions caused by alkylation damage, and it has clinical relevance: methylation of the MGMT promoter in glioblastoma tumors predicts a favorable response to the chemotherapy drug temozolomide.
III. Base Excision Repair (BER)
Base excision repair handles small, non-helix-distorting base lesions such as oxidized bases (8-oxoguanine), deaminated bases (uracil from cytosine deamination), and alkylated bases. The process begins when a DNA glycosylase recognizes and removes the damaged base by cleaving the glycosidic bond, creating an AP (apurinic/apyrimidinic) site. Multiple glycosylases exist, each specific for a particular type of lesion: uracil-DNA glycosylase (UDG) removes uracil from DNA, while OGG1 removes 8-oxoguanine. Next, AP endonuclease (APE1) cleaves the phosphodiester backbone 5' to the AP site. DNA polymerase beta then fills the single-nucleotide gap and uses its deoxyribose phosphate lyase activity to remove the remaining sugar-phosphate remnant. Finally, DNA ligase III/XRCC1 seals the nick.
This short-patch BER pathway, which replaces a single nucleotide, is the most common form. A long-patch variant replaces 2-10 nucleotides and uses Pol delta/epsilon, FEN1, and ligase I. Defects in BER are associated with increased cancer risk.
IV. Nucleotide Excision Repair (NER)
Nucleotide excision repair handles bulky, helix-distorting lesions such as pyrimidine dimers from UV damage, large chemical adducts like benzo[a]pyrene-DNA adducts, and intrastrand crosslinks. NER operates through two sub-pathways. Global Genome NER (GG-NER) scans the entire genome for damage, with the XPC-RAD23B complex recognizing helix distortion. Transcription-Coupled NER (TC-NER) repairs damage specifically on the transcribed strand of active genes: when RNA polymerase stalls at a lesion, the CSA and CSB proteins are recruited, ensuring faster repair of active genes.
After damage recognition, both sub-pathways converge on a shared mechanism. The TFIIH complex, which contains the XPB and XPD helicases, unwinds DNA around the lesion to create a bubble of approximately 25-30 base pairs. XPA verifies the damage while RPA stabilizes the resulting single-stranded DNA. Two endonucleases then make incisions on either side of the lesion: XPF-ERCC1 cuts on the 5' side and XPG cuts on the 3' side. The resulting oligonucleotide of approximately 24-32 nucleotides containing the damage is excised and released. DNA polymerase delta/epsilon fills the gap using the undamaged strand as a template, and DNA ligase I seals the nick.
Xeroderma pigmentosum (XP) is an autosomal recessive disorder caused by defects in any of the NER genes (XPA through XPG). Affected individuals display extreme sensitivity to UV radiation, a greater than 1,000-fold increased risk of skin cancer, and neurological degeneration in some forms. Cockayne syndrome, caused by defects in TC-NER (CSA or CSB genes), produces growth failure, neurological dysfunction, and photosensitivity but, interestingly, does not carry an increased cancer risk.
<image>Panel A: Step-by-step diagram of nucleotide excision repair (NER) showing damage recognition (XPC for GG-NER, stalled RNA Pol II for TC-NER), unwinding by TFIIH, verification by XPA, dual incision by XPF and XPG (with 5' and 3' cut sites marked), excision of the ~25-30 nt fragment, gap filling by Pol delta, and ligation. Panel B: Comparison diagram of BER and NER side by side, highlighting the key differences — BER removes a single damaged base via glycosylase creating an AP site, while NER excises a 24-32 nt oligonucleotide containing the bulky lesion. Panel C: Clinical photos-style illustration of xeroderma pigmentosum features (freckling, photosensitivity, skin lesions on sun-exposed areas) with a diagram showing the NER pathway with XP complementation groups (XPA through XPG) and which step each is involved in.</image>
V. Mismatch Repair (MMR)
Mismatch repair corrects base-base mismatches and small insertion/deletion loops (IDLs) that escape proofreading by DNA polymerase, and it is critical for maintaining microsatellite stability.
In E. coli, the MutHLS system operates as follows: MutS scans along DNA and recognizes the mismatch, then recruits MutL to form a MutS-MutL complex. MutH, an endonuclease, then cleaves the unmethylated (newly synthesized) strand at a hemimethylated GATC site. Strand discrimination relies on Dam methylase, which methylates adenine in GATC sites; the newly synthesized strand is temporarily unmethylated and thus identifiable as the error-containing strand. Helicase (UvrD) and exonucleases then remove the segment containing the error, and Pol III fills the gap while ligase seals the nick.
In eukaryotes, MSH2-MSH6 (MutS-alpha) recognizes single base mismatches and small IDLs of 1-2 nucleotides, while MSH2-MSH3 (MutS-beta) recognizes larger IDLs. MLH1-PMS2 (MutL-alpha) provides endonuclease activity for strand incision. Strand discrimination likely involves strand breaks such as Okazaki fragment gaps on the lagging strand or the 3' end on the leading strand, with PCNA and RFC helping to direct repair to the newly synthesized strand.
Lynch syndrome (Hereditary Nonpolyposis Colorectal Cancer, HNPCC) is an autosomal dominant disorder caused by germline defects in MMR genes, most commonly MLH1, MSH2, MSH6, or PMS2. Affected individuals face a 70-80% lifetime risk of colorectal cancer along with elevated risks of endometrial, ovarian, and gastric cancers. The hallmark of Lynch syndrome tumors is microsatellite instability (MSI), characterized by expansions and contractions of microsatellite repeats in tumor DNA. Clinical diagnosis relies on the Amsterdam criteria and Bethesda guidelines.
VI. Double-Strand Break (DSB) Repair
Double-strand breaks are the most dangerous type of DNA damage because both strands of the helix are severed simultaneously. They can result from ionizing radiation, oxidative stress, replication fork collapse, or certain chemicals. If unrepaired, DSBs lead to chromosome loss, translocations, or cell death. Two major pathways handle their repair.
Homologous Recombination (HR) uses a homologous template, typically the sister chromatid, for accurate repair and is primarily active during S and G2 phases when the sister chromatid is available. The process begins with end resection by the MRN complex (MRE11-RAD50-NBS1) and CtIP, which generate 3' single-stranded overhangs. RPA initially coats the single-stranded DNA, then is replaced by RAD51 with the help of BRCA2. The RAD51-coated filament invades the homologous template, forming a D-loop. DNA synthesis extends the invading strand using the template, and Holliday junctions are resolved to restore intact chromosomes. BRCA1 and BRCA2 are critical for HR, and mutations in these genes impair homologous recombination and predispose to breast and ovarian cancer.
Non-Homologous End Joining (NHEJ) directly ligates broken ends without requiring a homologous template. It is active throughout the cell cycle and predominates in G1. Although faster than HR, NHEJ is error-prone and can introduce small insertions or deletions at the junction. The process involves the Ku70/Ku80 heterodimer binding to the broken DNA ends, recruitment of DNA-PKcs (which tethers the two ends through its kinase activity), end processing by the Artemis nuclease, gap filling by DNA polymerases mu and lambda, and ligation by the XRCC4-Ligase IV complex. Defects in NHEJ components cause severe combined immunodeficiency (SCID) and radiosensitivity.
<image>Panel A: Side-by-side comparison of homologous recombination (HR) and non-homologous end joining (NHEJ) repair of a double-strand break. HR pathway shows end resection, RAD51 filament formation, strand invasion of sister chromatid, D-loop, DNA synthesis, and resolution. NHEJ pathway shows Ku70/80 binding, DNA-PKcs recruitment, end processing, and ligation. Panel B: Diagram illustrating the mismatch repair pathway in E. coli showing MutS recognizing a G-T mismatch, MutL and MutH recruitment, strand discrimination by hemimethylation at GATC sites, excision of the error-containing strand, and gap filling. Panel C: Flowchart showing DSB repair pathway choice based on cell cycle phase — NHEJ dominant in G1, HR dominant in S/G2 when sister chromatid is available.</image>
VII. Translesion Synthesis (TLS)
Translesion synthesis is a damage tolerance mechanism rather than a true repair pathway. When the replicative polymerase stalls at a lesion, specialized TLS polymerases from the Y-family take over. These include Pol eta, Pol iota, Pol kappa, and REV1. TLS polymerases lack proofreading activity and have low fidelity on undamaged DNA, but their larger, more open catalytic pockets can accommodate damaged bases.
Pol eta is particularly important because it can accurately bypass thymine dimers by inserting two adenines opposite a T-T dimer. Defects in Pol eta cause xeroderma pigmentosum variant (XP-V), which features normal NER but error-prone bypass of UV damage leading to skin cancer. TLS is coordinated by ubiquitination of PCNA at lysine 164: monoubiquitination by RAD6/RAD18 recruits TLS polymerases, while polyubiquitination activates a template switching pathway that is error-free. Although TLS is mutagenic, it allows cells to survive and complete replication when they otherwise could not.
VIII. The SOS Response in Bacteria
The SOS response is a global DNA damage response in E. coli triggered by extensive DNA damage. Under normal conditions, the LexA repressor binds to SOS box sequences upstream of approximately 40 SOS genes, keeping them repressed. When DNA damage occurs, single-stranded DNA accumulates at stalled replication forks, and RecA binds this ssDNA to form an activated RecA filament. RecA stimulates LexA autocleavage, which de-represses the SOS genes. The induced genes include DNA repair enzymes (such as uvrA and uvrB for NER), TLS polymerases (Pol IV/DinB and Pol V/UmuD'2C), and cell division inhibitors (sulA) that delay division to provide time for repair. The SOS response represents a last resort: it increases the mutation rate through error-prone TLS but allows bacterial survival under conditions that would otherwise be lethal.
