Calvin Yee Kim Guan Yee
Biomedical and Life Sciences (Bailrigg) | Year 3 | Degree: Bsc Biological Sciences
Uracil DNA glycosylase: An important tool for DNA repair in all living organisms
Uracil DNA glycosylase (UDG) is a DNA repair enzyme found in many organisms ranging from bacteria to humans as well as some viruses. UDGs have been well characterized in humans, bacteria, yeast and viruses but not particularly well in an intestinal parasite known as Giardia intestinalis. My project aims to understand how the UDG discovered in Giardia (GiUDG) compares to that of other well-described UDGs. By understanding how GiUDG works, a potential therapeutic target against Giardia may be developed. Furthermore, as Giardia is one of the earliest diverging eukaryotes, an understanding of its UDG could provide more information on the evolution of that particular enzyme.
GiUDG was first recombinantly expressed, purified and subsequently tested using an inhibition assay. Known inhibitors of UDGs (e.g. uracil and nifuroxazide) were mixed with GiUDG and the inhibition was measured by the ability of GiUDG to repair damaged DNA. Results showed that both the inhibitors were unable to inhibit GiUDG but rather, had enhanced its activity. This suggested that GiUDG may work differently from other UDGs, implying that a potential drug targeting Giardia is possible with more research.
Calvin Yee Kim Guan Yee
Biomedical and Life Sciences (Bailrigg) | Year 3 | Degree: Bsc Biological Sciences
Uracil DNA glycosylase: An important tool for DNA repair in all living organisms
All living organisms contain genes that carry genetic information in the form of a molecule known as DNA.
These genes make you who you are!
Unfortunately, the DNA is very vulnerable and there are many factors that can lead to the DNA being damaged.
One common example:
UV light exposure can damage DNA, causing skin cancer.
Our body has adapted unique mechanisms to repair DNA damage!
Repair mechanisms include:
1) Base Excision Repair (BER)
2) Nucleotide Excision Repair (NER)
3) Mismatch-specific repair (MMR)
4) Repair of DNA double-stranded breaks
This project aims to investigate the structure of a UDG found in Giardia intestinalis (G.intestinalis), a causative agent of giardiasis. An understanding of the structure of UDG in G.lamblia (GiUDG) would provide a potential therapeutic target for the treatment of giardiasis. In short, we can develop a drug to target and kill this parasite! Below are two short introductory videos to give you an idea of the BER pathway and the parasite.
Uracil DNA glycosylases of 6 different organisms.
DNA damage can come in many forms; one of which is the presence of a base called uracil on DNA. If uracil is present on the DNA, it has to be removed to avoid subsequent mutations.
The Base Excision Repair (BER) pathway is the primary repair pathway used to remove non-bulky DNA damage such as uracil.
Components of the BER pathway:
1) DNA glycosylases
2) AP endonuclease
3) DNA polymerase
4) DNA ligase
DNA glycosylases are enzymes that used to target and remove a specific base from DNA. Uracil DNA glycosylase (UDG) is a type of DNA glycosylase used to specifically eliminate uracil from DNA.
UDGs can be found in all living organisms. The image on the right shows the 3D structure of UDGs found in 6 different organisms.
They are quite similar in their overall structure except for that of the mimivirus.
By comparing the two structures, a disoriented loop region (pointed by black arrow) can be observed in the GiUDG model but not in human UDG. This loop region may be a potential divergent feature of GiUDG and can potentially be targeted in an attempt to treat giardiasis.
What is nifuroxazide?
- a drug that treats colitis and diarrhoea
- a proven inhibitor of human UDG
How does the structure of GiUDG compare to that of human UDG?
To answer the question above, we used an inhibitory approach whereby nifuroxazide was used to see whether or not it could block the action of GiUDG.
If nifuroxazide is able to inhibit GiUDG, it can be postulated that its structure would be fairly identical to that of human UDG due to a similar binding mechanism.
If nifuroxazide is unable to inhibit GiUDG, it can be postulated that its structure would be different from that of human UDG due to a different binding mechanism.
DNA repair assay showing the activity of GiUDG and E.coli UDG in 1) the absence of inhibitors, 2) the presence of nifuroxazide, 3) the presence of uracil.
Results
Methods
What did we find?
There was a cleaved band in the sample containing GiUDG + nifuroxazide, suggesting that GiUDG was still able to perform its function. Hence, nifuroxazide was unable to inhibit GiUDG.
Another interesting observation was that nifuroxazide had somehow enhanced the activity of GiUDG as the cleaved band was much more intense compared to the other GiUDG samples.
Now that we know that nifuroxazide cannot inhibit GiUDG, we can postulate that the structure of GiUDG is different from that of human UDG.
We then sought to predict the structure of GiUDG to allow comparison with human UDG.
Structure of human UDG
Model of the structure of GiUDG using human UDG as a template
Summary of findings
The 3D structure of GiUDG is clearly different from that of human UDG. This is evidenced by the presence of a disoriented loop region found in GiUDG but not found in human UDG. This suggested that GiUDG can be used as a potential therapeutic target for the treatment of giardiasis. If we can develop a drug that targets the loop region and inhibits GiUDG, we can eliminate the parasite and treat giardiasis.
Acknowledgements
I would like to thank my family and friends for their continuous support for me in completing my dissertation. I would also like to especially acknowledge my supervisor, Dr Sarah Allinson, for her tremendous support and guidance throughout my dissertation journey.
Conclusion
Procedure:
1) Mix GiUDG with nifuroxazide
2) Add substrate (uracil-containing DNA)
3) Check for activity using DNA repair assay (presence of a cleaved band indicates activity)
Future directions
In this study, we have only managed to predict the structure of GiUDG by generating a model using human UDG as a template. Future studies should focus on investigating the crystal structure of GiUDG in order to fully determine its 3D structure.
References
Evans, M. D., Dizdaroglu, M. & Cooke, M. S. (2004) Oxidative DNA damage and disease: induction, repair and significance. Mutation Research-Reviews in Mutation Research, 567(1), 1-61.
H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne.
(2000) The Protein Data Bank Nucleic Acids Research, 28: 235-242.
Krokan, H. E., Standal, R. & Slupphaug, G. (1997) DNA glycosylases in the base excision repair of DNA. Biochemical Journal, 325, 1-16.
Li, Guodong, Adam Henry, Stuart, Liu, Hao., Kang, Tian-Shu., Nao, Sang-Cuo., Zhao, Yichao., Wu, Chun., Jin, Jianwen., Zhang, Jia-Tong et al. (2020) “A Robust Photoluminescence Screening Assay Identifies Uracil-DNA Glycosylase Inhibitors against Prostate Cancer.” Chemical Science, vol. 11, no. 7, 2020, pp. 1750–1760.
Prorok, P., Alili, D., Saint-Pierre, C., Gasparutto, D., Zharkov, D. O., Ishchenko, A. A., Tudek, B. & Saparbaev, M. K. (2013) Uracil in duplex DNA is a substrate for the nucleotide incision repair pathway in human cells. Proceedings of the National Academy of Sciences of the United States of America, 110(39), E3695-E3703.
Sancar, A., Lindsey-Boltz, L. A., Unsal-Kacmaz, K. & Linn, S. (2004) Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annual Review of Biochemistry, 73, 39-85.
Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F.T., de Beer, T.A.P., Rempfer, C., Bordoli, L., Lepore, R., Schwede, T. (2018) SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46, W296-W303.
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