In genetics, crosslinking of DNA occurs when various exogenous or endogenous agents react with two nucleotides of DNA, forming a covalent linkage between them. This crosslink can occur within the same strand (intrastrand) or between opposite strands of double-stranded DNA (interstrand). These adducts interfere with cellular metabolism, such as DNA replication and transcription, triggering cell death. These crosslinks can, however, be repaired through excision or recombination pathways.
DNA crosslinking also has useful merit in chemotherapy and targeting cancerous cells for apoptosis, as well as in understanding how proteins interact with DNA.
Crosslinking agents
Many characterized crosslinking agents have two independently reactive groups within the same molecule, each of which is able to bind with a nucleotide residue of DNA. These agents are separated based upon their source of origin and labeled either as exogenous or endogenous. Exogenous crosslinking agents are chemicals and compounds, both natural and synthetic, that stem from environmental exposures such as pharmaceuticals and cigarette smoke or automotive exhaust. Endogenous crosslinking agents are compounds and metabolites that are introduced from cellular or biochemical pathways within a cell or organism.
Exogenous agents
Nitrogen mustards are exogenous alkylating agents which react with the N7 position of guanine. These compounds have a bis-(2-ethylchloro)amine core structure, with a variable R-group, with the two reactive functional groups serving to alkylate nucleobases and form a crosslink lesion. These agents most preferentially form a 1,3 5'-d(GNC) interstrand crosslink. The introduction of this agent slightly bends the DNA duplex to accommodate for the agent's presence within the helix. These agents are often introduced as a pharmaceutical and are used in cytotoxic chemotherapy. Cisplatin (cis-diamminedichloroplatinum(II)) and its derivatives mostly act on adjacent guanines at their N7 positions. The planar compound links to nucleobases through water displacement of one or both of its chloride groups, allowing cisplatin to form monoadducts to DNA or RNA, intrastrand DNA crosslinks, interstrand DNA crosslinks, and DNA-protein crosslinks. When cisplatin generates DNA crosslinks, it more frequently forms 1,2-intrastrand crosslinks (5'-GG), but also forms 1,3-intrastrand crosslinks (5-GNG) at lower percentages. When cisplatin forms interstrand crosslinks (5'-GC), there is a severe distortion to the DNA helix due to a shortened distance between guanines on opposite strands and a cytosine that is flipped out of the helix as a consequence of the GG interaction. Similar to nitrogen mustards, cisplatin is used frequently in chemotherapy treatment - especially for testicular and ovarian cancers. Chloro ethyl nitroso urea (CENU), specifically carmustine (BCNU), are crosslinking agents that are widely used in chemotherapy, particularly for brain tumors. These agents differ from other crosslinkers as they alkylate O6 of guanine to form an O6-ethanoguanine. This intermediate compound then leads to an interstrand crosslink between a GC basepair. These crosslinking agents only result in small distortions to the DNA helix due to the molecules' smaller size. Psoralens are natural compounds (furocoumarins) present in plants. These compounds intercalate into DNA at 5'-AT sequence sites and form thymidine adducts when activated in the presence of Ultra Violet-A (UV-A) rays. These covalent adducts are formed by linking the 3, 4 (pyrone) or 4', 5’ (furan) edge of psoralen to the 5, 6 double bond of thymine. Psoralens can form two types of monoadducts and one diadduct (an interstrand crosslink) with thymine. These adducts result in local distortions to DNA at the site of intercalation. Psoralens are used in the medical treatment of skin diseases, such as psoriasis and vitiligo. Mitomycin C (MMC) is from a class of antibiotics that are used broadly in chemotherapy, often with gastrointestinal related cancers. Mitomycin C can only act as a crosslinker when a DNA nucleotide has had a reduction to its quinone ring. When two dG's have been rearranged and methylated in this manner, a 5'-GC interstrand crosslink can be formed with the exo amines of each nucleobase. Mitomycin also harbors the ability to form monoadducts and intrastrand crosslinks with DNA as well. The interstrand crosslinks of Mitomycin C are formed in the minor groove of DNA, inducing a moderate widening or stretching to the DNA helix in order to accommodate for the presence of the molecule within the two strands.