Projects per year
The research focus of the group is the mechanistic analysis of DNA recognition and cleavage by prokaryotic defence systems such as Restriction-Modification and CRISPR. These enzymes protect bacteria from bacteriophages and thus moderate horizontal gene transfer. In addition, they are also important as the basis of many lab tools for manipulating DNA, such as the emergent genome editing technologies.
We use a dual experimental approach to studying DNA-protein interactions – combining single molecule microscopy with ensemble biochemistry, the latter including millisecond time-resolution rapid-mixing fluorescence spectroscopy, molecular biology and protein chemistry. More recently we have established collaborations to extend our studies to human cell culture.
DNA cleavage mechanisms in Restriction-Modification
We have focussed our research efforts on Restriction-Modification enzymes that use ATP-dependent protein machines to evade virus infection, addressing how these "molecular motors" convert chemical energy into mechanical events that lead to DNA cleavage. We have been able to demonstrate alternative properties of the helicase-like motor domains of these enzymes, including dsDNA translocation or molecular switching. These activities allow the enzymes to interact with sites that are distant along a phage genome. We aim to understand the diversity of these mechanisms, and their potential fitness costs to the bacteria.
The CRISPR/Cas effector nucleases
The Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR) and the CRISPR-associated (cas) genes comprise an adaptive immune system in bacteria and archaea. Silencing of foreign nucleic acids by CRISPR/Cas systems relies on a small CRISPR RNA (crRNA), the latter derived by processing transcribed CRISPR repeat-spacer arrays. We have developed a single molecule assay that allows the crRNA-guided recognition of specific DNA sequences to be followed in real time. Understanding how CRISPR/Cas systems achieve specificity will be particularly important in the manipulation of these proteins as tools for genome surgery, where specificity is paramount.
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14/02/19 → 13/02/22
EPICut - Molecular mechanisms, evolutionary impacts and applications of prokaryotic epigenetic-targeted immune systems
1/08/18 → 31/07/23
1/03/18 → 31/01/20
ENDO-Pore: high-throughput linked-end mapping of single DNA cleavage events using nanopore sequencingTorres Montaguth, O. E., Cross, S. J., Ingram, K. W. A., Lee, L., Diffin, F. M. & Szczelkun, M. D., 20 Aug 2021, (E-pub ahead of print) In: Nucleic Acids Research. 11 p., gkab727.
Research output: Contribution to journal › Article (Academic Journal) › peer-reviewOpen AccessFile17 Downloads (Pure)
5′ modifications to CRISPR Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavageMullally, G. E. L., Van Aelst, K., Mubarak Naqvi, M., Diffin, F. M., Karvelis, T., Gasiunas, G., Siksnys, V. & Szczelkun, M. D., 4 Jun 2020, In: Nucleic Acids Research. 13 p., gkaa477.
Research output: Contribution to journal › Article (Academic Journal) › peer-reviewOpen AccessFile36 Downloads (Pure)
Dimitriu, T., Szczelkun, M. D. & Westra, E. R., 5 Oct 2020, (E-pub ahead of print) In: Current Biology. 30, 19, p. R1189-R1202
Research output: Contribution to journal › Book/Film/Article review (Academic Journal) › peer-reviewOpen AccessFile
Szczelkun, Mark D (Recipient), 2004
Prize: Prizes, Medals, Awards and Grants