Harnessing promiscuous DNA-modification dependent enzymes used in phage defence
Lead Research Organisation:
Durham University
Department Name: Biosciences
Abstract
Background: Bacteriophages (phages) outnumber bacteria by ten to one, and the selection pressure has led to the evolution of phage-resistance systems that protect bacteria from phage predation. Many of these systems have proved invaluable to biochemists: restriction-modification and CRISPR-cas underpin the recombinant DNA and genome editing revolutions. This project will investigate the continuing attack and counter-attack between phage and bacteria.
We have recently characterised BrxU, a highly promiscuous restriction enzyme that recognises multiple different DNA modifications, and uses any nucleotide together with a wide selection of metals, to cleave modified DNA. The details of how these ligands are used, and how modification recognition leads to cleavage, remain to be uncovered. Furthermore, phages have evolved protein inhibitors of the BrxU family and exploring these interactions will allow further understanding of the BrxU mechanism. Beyond this fundamental biochemistry, the BrxU enzymes have biotechnological potential due to their ability to recognise cytosine DNA modifications that are present in up to 4% of the human genome. These epigenetic markers have roles in developmental processes, pluripotency of stem cells, neurodegenerative diseases and tumourigenesis. BrxU could therefore be used to map DNA modification sites in combination with next generation sequencing (NGS). This will provide a platform to better understand the role of epigenetic markers in developmental and disease processes.
Aims: This joint academic-industrial proposal will use molecular biology, microbiology, genomics, biochemistry and structural biology to investigate BrxU. The student will (1) examine BrxU biochemistry, namely (i) sequence specificity, (ii) DNA-modification specificity and (iii) inhibition of BrxU by phage protein IPI; (2) perform BrxU structural studies to understand DNA-modification recognition and inhibition by IPI; (3) apply BrxU to map DNA modifications via NGS.
We have recently characterised BrxU, a highly promiscuous restriction enzyme that recognises multiple different DNA modifications, and uses any nucleotide together with a wide selection of metals, to cleave modified DNA. The details of how these ligands are used, and how modification recognition leads to cleavage, remain to be uncovered. Furthermore, phages have evolved protein inhibitors of the BrxU family and exploring these interactions will allow further understanding of the BrxU mechanism. Beyond this fundamental biochemistry, the BrxU enzymes have biotechnological potential due to their ability to recognise cytosine DNA modifications that are present in up to 4% of the human genome. These epigenetic markers have roles in developmental processes, pluripotency of stem cells, neurodegenerative diseases and tumourigenesis. BrxU could therefore be used to map DNA modification sites in combination with next generation sequencing (NGS). This will provide a platform to better understand the role of epigenetic markers in developmental and disease processes.
Aims: This joint academic-industrial proposal will use molecular biology, microbiology, genomics, biochemistry and structural biology to investigate BrxU. The student will (1) examine BrxU biochemistry, namely (i) sequence specificity, (ii) DNA-modification specificity and (iii) inhibition of BrxU by phage protein IPI; (2) perform BrxU structural studies to understand DNA-modification recognition and inhibition by IPI; (3) apply BrxU to map DNA modifications via NGS.
Organisations
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/T008695/1 | 01/10/2020 | 30/09/2028 | |||
| 2713800 | Studentship | BB/T008695/1 | 01/10/2022 | 30/09/2026 |