In the cellular environment, DNA is under a constant threat of getting involved in a chemical reaction. It can be either due to alkylation, oxidation, or deamination. These reactions can damage the DNA and cause mutagenesis, leading to various diseases.
The human genome is made of 3 billion bases, and every day trillions of mutations occur in our body.
With this in mind, the question arises: why aren’t we all walking like diseased bodies?
Well, this is because of the repair mechanisms in our body, which correct the mutations and prevent the occurrence of diseases. One such repair pathway is base excision repair (BER). Most DNA damages are repaired by this pathway.
The enzyme that initiates the BER pathway is DNA glycosylase. It recognizes the DNA base lesions in the DNA and removes them, leaving the sugar-phosphate backbone intact. This creates an apurinic/apyrimidinic site that is called the AP site.
The abasic site is then repaired, and the original sequences are restored by enzymes like DNA polymerase, AP endonuclease, and DNA ligase.
Glycosylases were first discovered in bacteria, and Tomas Lindahl was the scientist who first observed uracil repair in DNA. Since then, glycosylases have been found in almost all living organisms, and H. E. Krokan was one of the scientists who extensively studied the enzyme.
In this article, we will cover the types of DNA glycosylases, how it works, and their lab applications.
Based on their crystal structures, DNA glycosylases have been categorized into four superfamilies:
Uracil-DNA glycosylase activities identified in mammalian cells are of four types based on cellular localization and substrate specificity. It includes:
In human cells, the major enzymes responsible for U:G mispairs are SMUG1 and TDG.
During the reaction, MYH produces the perfect substrate for OGG1, which corrects damaged bases (oxidized guanine bases) before it’s mispaired with adenine by replicative DNA polymerase during DNA replication. Thus, even during the disruption of MYH, no mutator phenotype is observed
Different glycosylases have different action mechanisms based on their structural characterizations. However, most glycosylases prefer the base-flipping mechanism (base excision repair). In this process, they flip out the damaged base or nucleotide residue from the DNA helix to the active site pocket for their excision and removal by cleaving base–sugar (N-glycosyl) bond.
The enzymes either use an activated water molecule or an amine residue, forming a Schiff base intermediate, to attack the damaged base.
The other mechanism is known as nucleotide excision repair. In this process, the structure-specific endonuclease recognizes structural distortions in the DNA and cuts the DNA on both sides of the lesion. This releases an oligonucleotide fragment containing the damaged site, which is then repaired by following the mechanism similar to base excision repair. The gap is filled by the enzyme DNA polymerase and DNA ligase.
One similarity in the base and nucleotide excision repairs is that in both the processes, transcription-active genes (required during the RNA synthesis) are preferentially rapidly repaired.
DNA glycosylase has a spectrum of in vitro applications in molecular biology and biochem lab assays. The assay also involves other reagents such as reaction buffer, EDTA, template DNA, DTT, and glycerol.
DNA glycosylases are used to correct the damaged base pairs in DNA helix, such as A:G, G:U, or G:T mismatches that occur due to oxidation, deamination, or alkylation of bases. Moreover, it can also correct the base modifications, such as those that occur due to aberrant methylation.
In addition to acting as DNA repair enzymes, DNA glycosylases also have an in vivo role in the repression of gene silencing by active demethylation in N. tabacum, A. thaliana, and some other plants.
DNA glycosylase enzyme helps to recognize and excise the damaged base mispairs in the DNA helix. It’s essential to maintain genomic integrity in vivo or in vitro applications.
Recombinant DNA glycosylases are commercially prepared and expressed in E.coli (mostly) for in vitro applications. They have major roles in vitro mutagenesis experiments to introduce site-directed mutations through PCR amplification. Moreover, it is also used to label oligonucleotide probes and prevent carryover contamination in PCR.
DNA glycosylases are a crucial enzyme in organisms to prevent mutation and maintain genomic integrity. This function is utilized by lab scientists in studying organisms’ metabolic processes and clinical applications.
In biochemistry labs, DNA glycosylases are used in various assays, including cloning, site-directed mutagenesis, and removing mutations. The enzyme also helps understand the genes’ functions and many other biochemical processes.
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DNA glycosylases are a base excision repair enzyme. It recognizes and excises the damaged base, forming an apurinic/apyrimidinic (AP) or abasic site, which is then replaced by the correct nucleotide. In this process, the undamaged strand serves as the template for the synthesis of the new nucleotide.
DNA glycosylases are categorized in four superfamilies based on their structural characterization. However, most of the enzymes of the family follow similar action mechanisms.
The DNA glycosylases’ enzymatic activity has a myriad of applications in many life science lab assays, such as gene expression studies, site-directed mutagenesis, and PCR cloning.
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