The Constant Assault on DNA
The DNA within every cell of the human body is under relentless attack. Endogenous threats, such as reactive oxygen species generated as byproducts of normal cellular metabolism, spontaneously hydrolyze nucleotides. Exogenous threats include ultraviolet (UV) radiation from the sun, which can cause adjacent thymine bases to fuse together; ionizing radiation from medical imaging or natural background sources, which can sever the sugar-phosphate backbone; and a multitude of chemical carcinogens found in tobacco smoke, charred food, and environmental pollutants. These insults can result in tens of thousands of individual molecular lesions per cell per day. Left unrepaired, this damage leads to mutations, which can disrupt essential genes, cause cellular dysfunction, and initiate the process of carcinogenesis. The stability of the genome, and by extension the viability of the organism, is entirely dependent on a sophisticated network of DNA repair mechanisms that have evolved to correct this damage with remarkable precision.
Direct Reversal: The Simplest Form of Repair
Not all repair requires excision and resynthesis. Direct reversal mechanisms are the most straightforward and energetically efficient repair pathways, correcting lesions by chemically reversing the damage in a single-step reaction.
Photoreactivation: A classic example, found in many bacteria, plants, and some animals (though not in placental mammals like humans), is the direct reversal of cyclobutane pyrimidine dimers (CPDs). These lesions, caused by UV radiation, are repaired by the enzyme photolyase. This enzyme captures energy from visible light and uses it to break the aberrant chemical bonds that link the pyrimidine bases, restoring them to their original state.
Alkyltransferase-mediated Repair: A highly relevant direct reversal pathway in humans deals with alkylation damage. Chemicals like methylnitrosourea can add alkyl groups (e.g., methyl or ethyl) to bases, most commonly to the O⁶ position of guanine. This alteration causes guanine to mispair with thymine during replication, leading to a point mutation. The enzyme O⁶-methylguanine-DNA methyltransferase (MGMT) directly addresses this problem. It catalyzes the transfer of the alkyl group from the damaged guanine to a cysteine residue within its own active site. This act of self-sacrifice permanently inactivates the MGMT molecule, which is then degraded. The consumption of MGMT means the cell’s capacity for this repair is limited and depends on new protein synthesis.
Base Excision Repair (BER): Correcting Small, Non-Helix-Distorting Lesions
Base Excision Repair is the primary pathway for correcting small, chemical alterations to individual DNA bases that do not significantly distort the double helix. These include oxidized bases (e.g., 8-oxoguanine), deaminated bases (e.g., uracil, which results from the deamination of cytosine), and alkylated bases.
The process is initiated by a class of enzymes called DNA glycosylases. Each glycosylase is highly specific for a particular type of base damage. For example, uracil-DNA glycosylase recognizes and removes uracil that has been misincorporated or formed by deamination. The glycosylase does not cut the DNA backbone; instead, it hydrolyzes the glycosidic bond between the damaged base and the deoxyribose sugar, creating an apurinic/apyrimidinic (AP) site—a location where a base is missing.
This AP site is then recognized by an AP endonuclease, which cleaves the phosphodiester backbone 5′ to the abasic site. In the predominant repair pathway, known as short-patch BER, DNA polymerase beta (in mammals) removes the leftover sugar-phosphate remnant, adds a single correct nucleotide, and then DNA ligase III, in complex with its stabilizing partner XRCC1, seals the nick. For more complex damage, a long-patch BER pathway can be employed, where a patch of 2-10 nucleotides is replaced.
Nucleotide Excision Repair (NER): Excising Bulky, Helix-Distorting Lesions
Nucleotide Excision Repair is a versatile, cut-and-patch mechanism designed to remove a wide array of bulky, helix-distorting lesions that disrupt the normal structure of DNA. These lesions, which include cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts caused by UV light, as well as large chemical adducts, are too substantial for BER to handle. NER operates through two distinct sub-pathways:
Global Genomic NER (GG-NER): This pathway surveys the entire genome for distortions in the DNA helix, independent of transcription. It is initiated by protein complexes that sense the DNA distortion. In humans, the XPC protein complex is a critical initial damage sensor. For some lesions, like CPDs, the DDB1-DDB2 (XPE) complex assists in recognition. Once the damage is identified, a multi-protein machinery is assembled to verify the lesion and unwind the DNA helix around it.
Transcription-Coupled NER (TC-NER): This pathway specifically targets lesions that block the progression of RNA polymerase II along the template strand of actively transcribed genes. The stalled polymerase itself serves as the damage signal. Specific proteins, such as CSA and CSB in humans, recognize the stalled complex and recruit the NER machinery. TC-NER is crucial because it prioritizes the repair of genes that are essential for current cellular function, preventing transcription arrest.
Following damage recognition in both sub-pathways, the core NER machinery is assembled. The helicase activity of the TFIIH complex unwinds the DNA around the lesion. Two endonucleases, XPG and the ERCC1-XPF complex, make precise incisions. XPG cuts the damaged strand 3′ to the lesion, while ERCC1-XPF cuts 5′ to the lesion, excising an oligonucleotide fragment approximately 24-32 nucleotides in length. The resulting single-stranded gap is then filled in by DNA polymerases (delta/epsilon) using the undamaged strand as a template, and DNA ligase seals the final nick.
Mismatch Repair (MMR): Correcting Replication Errors
Despite the exceptional proofreading ability of DNA polymerases, errors occasionally occur during DNA replication, leading to base-base mismatches (e.g., G paired with T) or small insertion-deletion loops (IDLs) that can arise from strand slippage, particularly in repetitive sequences called microsatellites. The Mismatch Repair system acts as a crucial post-replication spell-checker, improving replication fidelity by 100 to 1000-fold.
A key to MMR’s effectiveness is its ability to distinguish the newly synthesized, error-containing strand from the original template strand. In E. coli, this is achieved by the transient lack of methylation on the new strand at adenine residues within GATC sequences. The MutS protein homodimer recognizes and binds to the mismatch. It then recruits MutL, which activates MutH, an endonuclease that nicks the unmethylated daughter strand. Exonucleases degrade the nicked strand from the incision point past the mismatch, and the gap is resynthesized.
In humans, the process is more complex but conceptually similar. The strand discrimination signal is thought to involve nicks in the nascent DNA strand. The homologous proteins hMutSα (a heterodimer of MSH2 and MSH6) recognizes base-base mismatches and small IDLs, while hMutSβ (MSH2-MSH3) recognizes larger IDLs. hMutS recruits hMutLα (a heterodimer of MLH1 and PMS2), which coordinates the excision of the error-containing segment. Exonucleases (e.g., EXO1) remove the DNA, and the gap is filled by DNA polymerase delta and sealed by DNA ligase. Defects in MMR genes are strongly associated with hereditary nonpolyposis colorectal cancer (Lynch syndrome).
Double-Strand Break Repair: Handling the Most Dangerous Lesions
DNA double-strand breaks (DSBs) are among the most cytotoxic forms of DNA damage because they completely sever the chromosome. If left unrepaired, they can lead to large-scale genomic rearrangements, cell death, or cancer. Cells employ two main pathways to repair DSBs, which operate in a competitive manner.
Homologous Recombination (HR): HR is an error-free repair pathway that operates primarily during the S and G2 phases of the cell cycle, after DNA replication has provided a sister chromatid that can serve as a perfect template for repair. The process begins with resection, where the 5′ ends at the break are chewed back by nucleases to create 3′ single-stranded DNA overhangs. These overhangs are rapidly coated by the protein RPA to prevent degradation. The central recombinase enzyme, Rad51, then displaces RPA and forms a nucleoprotein filament that invades the homologous sequence on the sister chromatid. Using the sister chromatid as a blueprint, DNA synthesis extends the 3′ end to copy the missing genetic information. The resulting structures, called Holliday junctions, are ultimately resolved to produce two intact, error-free DNA molecules. Key proteins in human HR include BRCA1 and BRCA2; inherited mutations in these genes significantly increase the risk of breast, ovarian, and other cancers.
Non-Homologous End Joining (NHEJ): NHEJ is an error-prone but rapid repair pathway that can function throughout the cell cycle. It is the dominant pathway in mammalian cells. NHEJ directly ligates the two broken ends back together without the requirement for a homologous template. The Ku70/Ku80 heterodimer binds to the DNA ends first, protecting them and recruiting the core NHEJ machinery, including the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The ends are processed by various nucleases and polymerases to make them compatible for ligation, which is performed by DNA ligase IV in complex with XRCC4. Because NHEJ does not use a template, it often results in small insertions or deletions (indels) at the break site. While potentially mutagenic, NHEJ is essential for repairing breaks in non-cycling cells and during V(D)J recombination in the immune system to generate antibody diversity.
Interstrand Crosslink Repair: A Complex Challenge
Interstrand crosslinks (ICLs) are particularly severe lesions where a covalent bond forms between bases on opposite strands of the DNA double helix, physically preventing strand separation. This blocks both replication and transcription. ICLs are caused by certain chemotherapeutic agents (e.g., cisplatin, mitomycin C) and endogenous aldehydes.
Repair of ICLs is complex and requires the coordinated action of multiple repair pathways, including NER and HR. A specialized pathway, often activated during DNA replication when a replication fork stalls at an ICL, is called Fanconi Anemia pathway. Proteins in this pathway (FANCA, FANCD2, etc.) are recruited to the lesion and promote the unhooking of the crosslink, which involves incisions on both strands of the DNA by nucleases. This creates a DSB that can be repaired by HR, while the unhooked crosslinked nucleotide is dealt with by transfusion synthesis polymerases that can bypass the damage. Defects in the Fanconi Anemia pathway lead to the disease Fanconi anemia, characterized by bone marrow failure, developmental abnormalities, and a high predisposition to cancer.
The Consequences of Repair Failure
When DNA repair systems fail, the consequences are severe and directly linked to human disease. The accumulation of unrepaired DNA damage is a fundamental cause of cancer. For instance, defects in NER cause Xeroderma Pigmentosum (XP), characterized by extreme sensitivity to UV light and a greater than 10,000-fold increased risk of skin cancer. As noted, MMR defects cause Lynch syndrome, and HR defects (e.g., in BRCA1/2) lead to hereditary breast and ovarian cancer syndromes. Beyond cancer, defective DNA repair is implicated in neurodegenerative diseases like Alzheimer’s and Parkinson’s, and it is a primary driver of the aging process itself, as the gradual accumulation of mutations and cellular damage leads to tissue dysfunction and organismal decline. The study of DNA repair is therefore not only a field of basic biological interest but also a critical area for therapeutic development, as evidenced by the success of PARP inhibitors in treating cancers with HR deficiencies.