Maintenance of bacterial genome stability

Many bacterial species spend much of their life cycle in stationary phase during which only a single copy of the chromosome is present. In many of these cases the bacterium is also endowed with a two-components nonhomologous end-joining (NHEJ) system that mends double strand breaks (DSBs) through the direct joining of the DNA ends. Bacterial NHEJ is composed of the homodimer Ku and DNA ligase D (LigD), a multifunctional protein with polymerization, ligase and nucleolytic activities. Bacillus subtilis is a Gram⁺ spore-forming bacterium with a NHEJ system constituted by Ku (BsuKu) and LigD (BsuLigD) and whose genes are expressed in the developing spore. Deletion of those genes sensitizes B. subtilis cells to ionizing radiation in the stationary phase and their spores to several DNA-damaging treatments that cause DSBs. Our previous results showed that BsuKu interacts functionally with and stimulates BsuLigD enabling it to generate synaptic intermediates to repair DSBs through the coordinated action of the polymerization and ligase activities. Our recent results have shown an unforeseen ability of BsuLigD to cleave apurinici/apyrimidinic (AP) sites through its polymerization domain (PolDom) site, specifically when those AP sites are proximal to recessive 5’-ends, coordinating such an incision with further nucleotide insertion and final ligation steps.

With this project, it is intended to get further insights into the bacterial NHEJ system using BsuKu, BsuLigD, and the homologous proteins from the Gram negative bacterium Pseudomonas aeruginosa PaeKu, and PaeLigD as model proteins.

To get a better understanding of the bacterial NHEJ mechanism:

-It will be studied, by means of the set up of in vitro assays, the repair of synthetic DNA ends that mimic DSBs to infer the requisites necessary to coordinate those activities.

-Additionally, it will be studied whether there is a convergent evolution regarding the ability to process internal AP sites proximal to recessive ends between the bacterial PolDom and the  AP processing activities of bacterial PolDom are also present in the eukaryotic functional homolog NHEJ DNA polymerase lambda.