How bacteria replicate their DNA in spite of barriers, one molecule at a time

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Activities of DNA polymerases in replication result in collisions which, if remaining unresolved, can be lethal and therefore must be resolved efficiently. Extensive knowledge exists from genetics and biochemistry about the enzymes involved, but we know little of how this is achieved at the level of single molecules. Here we integrate interdisciplinary expertise, using E. coli as a model to study replication collisions and subsequent crucial repair, modifying cells to label key proteins used in replication used in replication and repair. We will use artificial blocks to controllably mimic collisions both in vitro and in living cells and native blocks involving RNAP from transcription that will allow us to study collisions in defined areas of the chromosome in any orientation. Some replisomes remain following collisions while others disassemble. By using labelled replisomes and blocks probed with super-resolved single-molecule microscopy, microfluidics and novel AI biocomputation tools, we will be able to define the conditions when forks disassemble under physiological conditions. We will then visualise which repair proteins are recruited and, finally, be able to characterise how restart proteins can re-recruit active replisomes to continue synthesis. We will also search for hitherto undiscovered factors that assist in vital replication collision resolution.

Our analyses will address fundamental questions concerning the resolution of collisions between replication molecular machinery and nucleoprotein blocks. DNA replication and repair offer key antibiotic targets, and we anticipate our findings will have longer term societal benefit in addressing how poisons targeting these processes can be tolerated by cells and lead to antibacterial resistance, aiding development of new antibiotics in addition to substantive development of new microscopy instrumentation, bioinformatics and high-precision analytical software tools of wide benefit to the biosciences.