Project No.2431

STANDARD PROJECT

Primary Supervisor

Dr Jan Janouskovec – University of Southampton

Co-Supervisor(s)

Dr Anastasios Tsaousis – University of Kent

Dr Franklin Nobrega – University of Southampton

Summary

DNA polymerase enzymes are indispensable in replication and drive genome evolution through mutational pressure

Polymerases with outstanding characteristics, such as high and low fidelity, also have applications in genetic engineering and biotechnology. This makes DNA polymerases attractive targets for characterisation from novel resources.

One such resource are DNA polymerases that replicate chloroplast and mitochondrial genomes with unusual chromosomal architectures:

The extremophilic alga Cyanidinioschyzon merolae has a super-compacted chloroplast genome nearly devoid of intergenic regions. We hypothesize that this is due to an unusual deletion-prone activity of a DNA polymerase of bacterial origin. You will purify the enzyme from Cyanidinioschyzon, and heterologously express a codon harmonized version in Escherichia coli. This enzyme will be used in in vitro assays to characterize its mutation rates and sequence contexts that favour deletion. You will use Alphafold2 models to identify key residues that drive function, and resolve its structure by crystallography. Finally, you will generate an enzyme knockout strain in Cyanidioschyzon, and sequence chloroplast genomes from multiple wildtype strains, to study mutational accumulation over generations in vivo.

Several other eukaryotes have unique organellar genomes. For example, chloroplasts in ulvophyte algae contain hairpin chromosomes, and those in dinoflagellates have single-gene minicircles, possibly comprising RNA/DNA heteroduplexes. You will identify genes for organellar polymerases in these organisms based on phylogenetic analysis, targeting pre-sequences and available proteomic data. You will then heterologously express the genes in E. coli and characterize their kinetics as above. You will also use chloroplast and nuclear transformation systems (e.g., in dinoflagellates) to characterise polymerase function in vivo and describe replication origins of plasmid-like molecules in eukaryotes.

Your findings will improve our understanding of how polymerases direct genome evolution. We will also connect with industrial partners to consider some enzymes you find for biotechnological applications, such as deletion-prone polymerases for random mutagenesis.