Introduction
DNA glycosylases, an ancient family of DNA repair proteins, play a crucial role in removing uracil from DNA. The discovery of uracil N-glycosidase (Ung) in Escherichia coli, capable of cleaving uracil–deoxyribose bonds, marked a breakthrough. Subsequently, various DNA glycosylases were identified across species. Mammals possess eleven DNA glycosylases, categorized into four distinct superfamilies: uracil DNA glycosylases (UDGs), helix-hairpin-helix (HhH) glycosylases, 3-methyl-purine glycosylase (MPG), and endonuclease VIII-like (NEIL) glycosylases.
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Structure and Substrate Recognition
DNA glycosylases exhibit high specialization, each possessing a unique structure and substrate specificity. Despite this diversity, they share a common principle of action. These enzymes recognize damaged DNA bases by rotating them out of the DNA helix into a fitting pocket that harbors the active site. Steric exclusion and catalytic efficiency play key roles in achieving substrate selectivity. Upon successful base fitting, cleavage of the N-glycosidic bond occurs, resulting in the release of a free base and the generation of an AP-site in the DNA.
Detection and Verification of Base Damage
Detecting a single damaged base among regular bases poses a considerable challenge, considering that a human cell experiences around 10^4 base lesions per day. The mechanisms employed by DNA glycosylases for efficient damage detection are not fully elucidated, but biochemical and structural studies offer valuable insights. The concept of DNA scanning, facilitated by non-specific interactions, sliding along the DNA duplex, and scanning for irregular bases, is proposed. Recent observations suggest that DNA glycosylases harboring [4Fe4S] clusters may use charge transfer through the DNA for damage detection.
Damage pre-selection strategies involve the examination of the chemical surface of single bases, minimizing the effort required for damage detection. DNA breathing, influenced by base lesions affecting base pairing dynamics, likely facilitates base pre-scanning.
Formation of a Mature Enzyme-Substrate Complex
For final damage verification, the damaged base must be flipped out of the DNA helix and accommodated in the active site cavity of the glycosylase. UNG, a well-studied glycosylase, induces a bend in the DNA, stabilizes the double helix, and selectively excises uracil. Other glycosylases like TDG and OGG1 exhibit broad substrate specificities and utilize distinct mechanisms for damage recognition. OGG1, for instance, induces significant DNA bending upon recognizing an 8-oxoG substrate.
Fig. 1 DNA glycosylase structural superfamilies Brzozowski J. S., Skelding K. A. 2019).
Catalysis of Base Removal
DNA glycosylases are categorized into monofunctional and bifunctional enzymes based on their catalytic mechanisms. Monofunctional glycosylases, exemplified by UNG, perform base excision using an activated water molecule for nucleophilic attack on the N-glycosidic bond. Bifunctional glycosylases, like OGG1, use an amino group of a lysine side chain for base cleavage, coupling base excision with an AP-lyase step. The catalytic mechanisms can vary considerably within the glycosylase superfamily, reflecting the need for fine-tuning substrate specificity and catalytic efficiency.
AP-site Dissociation and Turnover of Glycosylases
Upon base release, DNA glycosylases tend to stay bound to the product of their action, the AP-site. The release of the glycosylase from the AP site is rate-limiting in the base excision repair (BER) process. This association with AP sites protects cells against their cytotoxic and mutagenic effects. Regulation of AP-site interaction involves the recruitment of downstream BER factors and posttranslational modifications. Phosphorylation and SUMO modification of glycosylases like UNG2 and TDG play roles in regulating their association with AP sites.
Functions Beyond DNA Repair
While DNA glycosylases are primarily involved in DNA repair, their structural and biochemical properties suggest a broader spectrum of genetic functions. They play essential roles in innate immunity and antibody diversification. In innate immunity, APOBEC3 family proteins induce cytosine deamination, leading to uracil excision by UNG2 and subsequent degradation of viral DNA. In antibody diversification, UNG2, AID, and APE1 are crucial for somatic hypermutation (SHM) and class switch recombination (CSR), processes vital for generating diverse antibody repertoires.
DNA Glycosylases in Immunity
APOBEC3 proteins, part of the innate immune system, inhibit retroviral replication by inducing cytosine deamination. UNG2 and APE1 collaborate with APOBEC3G to introduce uracil and cleave viral DNA, contributing to antiviral defense. In adaptive immunity, UNG2 plays a pivotal role in SHM and CSR. The coordinated action of UNG2, AID, and APE1 ensures targeted DNA cleavage, initiating CSR.
DNA Glycosylases in DNA Methylation Control
Active DNA demethylation, involving the enzymatic removal of 5-meC, has been linked to DNA glycosylases. In plants, bifunctional DNA glycosylases such as ROS1 and DME participate in targeted demethylation. In vertebrates, MBD4 and TDG are implicated in active demethylation. UNG2, while not directly excising 5-meC, may assist in targeted demethylation processes.
In conclusion, DNA glycosylases emerge as guardians of genomic integrity, orchestrating intricate processes of damage detection and repair. Their versatile functions extend beyond DNA repair, encompassing roles in immunity, antibody diversification, and DNA methylation control. Unraveling the complexities of DNA glycosylases enhances our understanding of genome maintenance mechanisms and opens avenues for potential therapeutic interventions in conditions involving DNA damage and epigenetic regulation.
References
- Brzozowski J. S., Skelding K. A. The multi-functional calcium/calmodulin stimulated protein kinase (CaMK) family: emerging targets for anti-cancer therapeutic intervention. Pharmaceuticals. 2019, 12(1): 8.
- Brooks S. C., et al. Recent advances in the structural mechanisms of DNA glycosylases. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics. 2013, 1834(1): 247-271.
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