Translesion Synthesis: How DNA Bypasses Tough Lesions

Cells constantly encounter DNA damage from environmental factors and normal metabolic processes. While most damage is repaired, some lesions stall replication, threatening genomic integrity. To overcome this, cells use translesion synthesis (TLS), a specialized mechanism that allows replication to continue despite obstacles.

TLS is essential for cell survival but comes with risks, as it can introduce mutations. Understanding how TLS functions clarifies its impact on genetic stability and disease development.

Mechanisms Of Translesion

When replication machinery encounters a damaged DNA template, high-fidelity polymerases struggle to proceed. These enzymes, which typically ensure precise base pairing, stall at lesions such as thymine dimers or abasic sites, risking replication fork collapse. To prevent this, cells activate TLS, allowing specialized polymerases to temporarily take over and bypass the obstruction. This switch is tightly regulated to minimize errors while ensuring replication continues.

A key regulatory step in TLS involves monoubiquitination of proliferating cell nuclear antigen (PCNA), a sliding clamp that coordinates DNA synthesis. When replication stalls, the E3 ubiquitin ligase RAD18 attaches a ubiquitin molecule to PCNA, altering its interactions with polymerases. This modification recruits TLS polymerases, which possess flexible active sites capable of accommodating distorted DNA structures. Unlike replicative polymerases, these enzymes can insert nucleotides opposite lesions, allowing synthesis to proceed past the damage.

TLS unfolds in two phases: nucleotide insertion and extension. In the first, a TLS polymerase incorporates a base opposite the lesion, often relying on Watson-Crick-like hydrogen bonding or alternative mechanisms such as Hoogsteen base pairing. The choice of polymerase depends on the lesion type; for example, DNA polymerase η efficiently bypasses UV-induced cyclobutane pyrimidine dimers, while polymerase κ specializes in bulky adducts. Once a nucleotide is inserted, a second TLS polymerase or the same enzyme extends the nascent strand until a high-fidelity polymerase resumes normal replication.

Specialized DNA Polymerases

Cells rely on distinct TLS polymerases, each adapted to bypass specific DNA damage. Unlike replicative polymerases, which maintain high fidelity through stringent base selection and proofreading, TLS polymerases operate with relaxed base-pairing rules and lack exonucleolytic proofreading. This trade-off allows them to accommodate damaged nucleotides but also increases mutation risk. The balance between damage tolerance and mutation risk depends on the polymerase, as different enzymes exhibit varying accuracy and lesion specificity.

DNA polymerase η (Pol η) is well-studied for its role in bypassing UV-induced cyclobutane pyrimidine dimers (CPDs). Mutations in the POLH gene, which encodes Pol η, cause xeroderma pigmentosum variant (XP-V), leading to extreme sun sensitivity and increased skin cancer risk. Structural studies reveal that Pol η’s spacious, solvent-exposed active site allows it to incorporate two adenines opposite a thymine dimer with minimal DNA distortion, making it one of the most accurate TLS polymerases.

In contrast, DNA polymerase κ (Pol κ) specializes in bypassing bulky adducts from environmental mutagens like benzo[a]pyrene diol epoxide (BPDE), a carcinogenic metabolite of tobacco smoke. Unlike Pol η, which primarily inserts nucleotides, Pol κ extends DNA synthesis beyond the lesion. Its active site contains an “N-clasp” structure that stabilizes the DNA substrate, allowing it to accommodate distorted templates and continue elongation after another TLS polymerase inserts a nucleotide.

DNA polymerase ι (Pol ι) exhibits an unusual preference for Hoogsteen base pairing, where the incoming nucleotide interacts with the template base at an altered angle. This mechanism enables Pol ι to bypass oxidative lesions like 8-oxo-guanine but results in frequent misincorporations, making it one of the least accurate TLS polymerases.

Pol ζ, a heterodimer composed of the catalytic subunit Rev3 and accessory subunit Rev7, plays a distinct role by extending mismatched or lesion-containing primer termini. Unlike other TLS polymerases, which act transiently, Pol ζ is essential for completing replication past certain damage types that could otherwise cause replication fork collapse. Studies in Pol ζ-deficient mouse models show its importance in embryonic development, as its absence leads to early lethality due to genomic instability.

Catalytic And Noncatalytic Functions

TLS polymerases are primarily recognized for their catalytic activity, enabling them to bypass DNA lesions that stall replication. Their structural flexibility influences efficiency and accuracy, as differences in active site architecture affect nucleotide selection. Some polymerases, such as Pol η, exhibit relatively high fidelity when bypassing specific lesions, while others, like Pol ι, frequently introduce errors.

Beyond nucleotide incorporation, TLS polymerases contribute to replication through noncatalytic functions that influence DNA damage response pathways. Some interact with replication and repair proteins independently of polymerase activity, acting as scaffolds or signaling mediators. For example, Pol ζ stabilizes replication forks under stress, with its Rev7 subunit participating in protein-protein interactions that regulate cell cycle progression and checkpoint activation.

TLS polymerases also interact with post-translational modifications like ubiquitination and sumoylation, which regulate recruitment and activity. Pol κ, for instance, associates with chromatin via its ubiquitin-binding domain, allowing it to function in damage tolerance pathways beyond its catalytic role. TLS polymerases thus contribute to genome maintenance not only by synthesizing DNA across lesions but also by coordinating broader repair and signaling networks.

Common Lesions Bypassed

DNA is constantly exposed to damaging agents that alter its structure and impede replication. Among the most common lesions are UV-induced cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts, both resulting from sun exposure. These distortions prevent proper base pairing, leading to replication stalling. Cells rely on TLS polymerases such as Pol η to insert adenines opposite CPDs, reducing mutagenesis. However, 6-4 photoproducts pose greater challenges due to their more pronounced helical distortion, often requiring multiple TLS polymerases for bypass.

Oxidative damage is another major source of replication-blocking lesions, particularly in metabolically active cells where reactive oxygen species (ROS) are generated. One prevalent oxidative modification, 8-oxo-guanine, mispairs with adenine, increasing the risk of G-to-T transversions. Pol ι and Pol κ contribute to bypassing this lesion, though their error-prone nature can introduce mutations. Cells deficient in these polymerases exhibit increased sensitivity to oxidative stress, highlighting their role in replication fidelity.

Bulky adducts from tobacco smoke carcinogens or chemotherapy agents like cisplatin further complicate replication. These lesions create significant steric hindrance, preventing high-fidelity polymerases from proceeding. Pol κ and Pol ζ often bypass such damage, with Pol κ facilitating insertion and Pol ζ extending the nascent strand. The efficiency of this process varies depending on the adduct’s chemical composition, influencing mutation rates and drug resistance in tumor cells.

Interactions With Other Repair Processes

TLS functions within a broader network of DNA repair pathways that maintain genome integrity. Its relationship with other repair mechanisms determines whether a lesion is bypassed, excised, or triggers further cellular responses. Because TLS polymerases lack proofreading activity and can introduce mutations, cells tightly regulate their activity.

Nucleotide excision repair (NER) removes bulky lesions that distort the DNA helix, such as UV-induced photoproducts and chemical adducts. When replication forks encounter these lesions before repair is complete, TLS polymerases can temporarily take over to prevent stalling. However, cells prefer NER to fully eliminate damage when possible, as TLS introduces permanent mutations. Defects in NER components, such as those seen in xeroderma pigmentosum, increase reliance on TLS polymerases, raising mutagenesis rates.

TLS also interacts with homologous recombination (HR), which resolves stalled replication forks and double-strand breaks. If a lesion is too structurally disruptive for TLS polymerases to bypass, replication forks may collapse, triggering HR-mediated fork restart. Some TLS polymerases, such as Pol ζ, facilitate this process by extending stalled DNA strands to generate recombination substrates. Excessive reliance on TLS in HR-deficient cells, such as those with BRCA1 mutations, has been linked to increased chromosomal instability.

Effects On Genetic Stability

While TLS allows cells to overcome replication-blocking lesions, its error-prone nature affects genetic stability. The relaxed base-pairing rules of TLS polymerases enable DNA synthesis across damaged sites, but they also increase the likelihood of incorporating incorrect nucleotides. The extent of mutagenesis depends on lesion type, the polymerase involved, and post-replicative repair mechanisms.

TLS-driven mutations vary in consequence depending on their genomic location. In coding regions, errors may result in missense or nonsense mutations that alter protein function, potentially contributing to disease. For example, cancer genomics studies identify TLS polymerases as a source of mutations in tumor suppressor genes, linking error-prone bypass to oncogenesis. In noncoding regions, TLS-driven mutations can affect regulatory elements, influencing gene expression and contributing to cellular dysfunction. The long-term effects of TLS on genetic stability are especially evident in aging cells, where cumulative mutations compromise genome integrity.