DNA Crosslinking: Pathways, Consequences, and Cancer

DNA crosslinking disrupts normal cellular processes by covalently linking DNA strands or attaching proteins to DNA. These lesions interfere with replication and transcription, making them particularly harmful if left unrepaired. Cells have evolved multiple repair mechanisms to address crosslinks, but when these fail, serious consequences arise.

Understanding how DNA crosslinks form, how cells attempt to fix them, and what happens when repair mechanisms break down is crucial for recognizing their role in diseases like cancer.

Types Of DNA Crosslinks

DNA crosslinking occurs in different ways, leading to distinct lesions that disrupt cellular function. These modifications involve chemical bonds forming between nucleotides on the same strand, between complementary strands, or between DNA and proteins. Each type presents unique challenges for replication and transcription, requiring specialized repair mechanisms to restore genomic stability.

Intrastrand

Intrastrand crosslinks form when two nucleotides on the same DNA strand become covalently bonded. These are often induced by ultraviolet (UV) radiation, platinum-based chemotherapeutics like cisplatin, or alkylating agents. A well-known example is the thymine dimer, where adjacent thymine bases form a covalent bond due to UV exposure. Such crosslinks distort the DNA helix, interfering with polymerase activity during replication and transcription. If left unresolved, they can lead to mutations, particularly in rapidly dividing cells.

The nucleotide excision repair (NER) pathway plays a central role in fixing these lesions by recognizing the distortion, excising the damaged region, and synthesizing a correct replacement using the undamaged strand as a template. Studies published in Nature Communications (2021) highlight how defects in NER contribute to diseases like xeroderma pigmentosum, where individuals exhibit extreme sensitivity to sunlight and a heightened risk of skin cancer due to ineffective repair of UV-induced crosslinks.

Interstrand

Interstrand crosslinks (ICLs) form when two complementary DNA strands are covalently linked, preventing strand separation. These lesions block essential processes such as replication and transcription, leading to stalled replication forks and potential chromosomal instability. ICLs can be caused by bifunctional alkylating agents, such as mitomycin C or nitrogen mustards, commonly used in chemotherapy.

Repairing ICLs requires coordination between multiple pathways, including the Fanconi anemia (FA) pathway, homologous recombination (HR), and translesion synthesis (TLS). Research published in Cell Reports (2022) demonstrated that mutations in FA genes impair ICL repair, contributing to Fanconi anemia, a disorder characterized by bone marrow failure and cancer predisposition. The inability to resolve ICLs efficiently can result in double-strand breaks, further elevating the risk of genomic instability and malignant transformation.

DNA–Protein

DNA–protein crosslinks (DPCs) occur when proteins become covalently attached to DNA, obstructing replication and transcription machinery. These lesions can arise from exposure to formaldehyde, reactive oxygen species, or metabolic byproducts. Topoisomerases, which relieve supercoiling during DNA processing, are prone to forming DPCs when trapped by inhibitors such as etoposide or camptothecin, both used in cancer treatment.

The proteasome and specialized repair enzymes like tyrosyl-DNA phosphodiesterase 1 (TDP1) and 2 (TDP2) play essential roles in resolving DPCs by degrading the crosslinked protein and restoring DNA integrity. A study in Molecular Cell (2023) found that defects in DPC repair contribute to neurodegenerative disorders and cancer, emphasizing the importance of efficient clearance of these lesions. Failure to resolve DPCs can lead to replication stress, genomic instability, and increased susceptibility to mutagenesis.

Factors Causing Crosslink Formation

DNA crosslinks arise from endogenous and exogenous sources, each contributing to genomic instability. Endogenous factors include metabolic byproducts such as reactive oxygen species (ROS) and aldehydes, which induce covalent bonding between DNA strands or between DNA and proteins. ROS, generated during normal cellular respiration, can oxidize nucleotide bases and initiate crosslinking reactions. Aldehydes, including formaldehyde and acetaldehyde, are produced during lipid peroxidation and alcohol metabolism and have been implicated in crosslink formation. A study in Nature Genetics (2020) demonstrated that deficiencies in aldehyde detoxification enzymes, such as ALDH2, lead to increased DNA crosslinking and genomic instability, particularly in individuals with alcohol intolerance.

Exogenous agents further exacerbate crosslink formation, particularly through environmental exposures and therapeutic interventions. UV radiation from sunlight induces intrastrand crosslinks, such as thymine dimers, by directly exciting nucleotide bases. Ionizing radiation, including X-rays and gamma rays, generates hydroxyl radicals that facilitate interstrand crosslinking. Chemical agents, such as platinum-based drugs like cisplatin and bifunctional alkylating agents like nitrogen mustards, introduce crosslinks as part of their cytotoxic action against cancer cells.

While these therapeutics effectively target rapidly dividing cells, they also damage normal tissues, leading to off-target toxicity. Research published in Cancer Research (2021) highlighted how cisplatin-induced crosslinks contribute to treatment resistance by triggering DNA repair pathways that allow cancer cells to survive.

Occupational and environmental exposures play a significant role in crosslink formation. Workers in industries involving formaldehyde, a known DNA–protein crosslinking agent, face increased risks of genomic damage. Chronic exposure has been linked to hematological malignancies. Similarly, exposure to polycyclic aromatic hydrocarbons (PAHs) from tobacco smoke, vehicle exhaust, and charred foods introduces reactive intermediates capable of forming crosslinks. A longitudinal study in Environmental Health Perspectives (2022) found that individuals with prolonged PAH exposure exhibited higher levels of DNA crosslinking, correlating with an elevated risk of lung and bladder cancers.

Repair Pathways Maintaining Genomic Integrity

Cells have developed multiple repair mechanisms to counteract DNA crosslinking, each tailored to address the unique structural challenges these lesions present. Standard repair processes such as base excision repair (BER) or mismatch repair (MMR) are insufficient. Instead, specialized pathways detect, excise, and replace damaged DNA while maintaining chromosomal stability.

The Fanconi anemia (FA) pathway orchestrates the repair of interstrand crosslinks (ICLs). Upon detection of an ICL, the FA core complex ubiquitinates FANCD2 and FANCI, signaling for nucleolytic incisions on either side of the lesion. This cleavage allows for translesion synthesis (TLS) polymerases to bypass the crosslink, temporarily enabling replication progression. Subsequent steps involve homologous recombination (HR), which utilizes a sister chromatid as a template to restore the DNA sequence. Mutations in FA pathway genes impair this process, predisposing individuals to bone marrow failure and malignancies.

For DNA–protein crosslinks (DPCs), repair requires a combination of proteolytic degradation and DNA processing enzymes. The proteasome degrades trapped proteins, particularly when topoisomerases become covalently linked to DNA. Tyrosyl-DNA phosphodiesterases (TDP1 and TDP2) further facilitate this process by cleaving the residual DNA-protein bond, allowing for subsequent repair synthesis. The importance of these enzymes is evident in neurological disorders such as spinocerebellar ataxia with axonal neuropathy (SCAN1), where TDP1 mutations lead to persistent DPC accumulation and progressive neurodegeneration.

Cellular Consequences Of Unrepaired Crosslinks

When DNA crosslinks persist without repair, they obstruct essential processes such as replication and transcription, triggering replication fork stalling and RNA polymerase arrest. Cells encountering crosslinks experience prolonged replication stress, activating checkpoint signaling pathways that delay the cell cycle to allow time for repair. If the lesion remains unresolved, cells may enter prolonged arrest, depleting resources and impairing proliferation.

Replication stress often results in double-strand breaks (DSBs), a hazardous form of genomic instability. When replication forks collapse at crosslink sites, they leave broken DNA ends that must be repaired through homologous recombination or non-homologous end joining (NHEJ). While homologous recombination restores the original sequence with high fidelity, NHEJ is more error-prone and may introduce insertions or deletions, increasing the likelihood of mutations. Over time, these errors can lead to chromosomal rearrangements and amplifications—alterations frequently observed in precancerous and malignant cells.

Association With Cancer

DNA crosslinking contributes to cancer by promoting genomic instability. When crosslinks interfere with replication and transcription, they create an environment where mutations accumulate, increasing the likelihood of oncogenic transformation. Tumor suppressor genes, such as TP53 and BRCA1/2, play significant roles in responding to DNA damage, but when these pathways are compromised, crosslink-induced mutations can drive malignant progression.

Research in Nature Cancer (2022) highlighted that BRCA-deficient tumors exhibit heightened sensitivity to crosslinking agents like cisplatin, as they rely on homologous recombination for repair. This vulnerability has been exploited in cancer therapy, particularly with PARP inhibitors, which further impair DNA repair and selectively kill tumor cells with defective repair mechanisms.