RESERACH SUMMARY

Our aim is to characterise the involvement of DNA repair and segregation in the stability of the genetic material in Firmicutes using two model systems.

Bacillus subtilis is used to study repair-by-recombination, and plasmid pSM19035 to study segregational stability.

In the first model, we showed that

i) during double-strand break repair, RecN in concert with polynucleotide phosphorylase (PNPase) promotes the dynamic recruitment of DNA ends. PNPase distributively removes few nucleotides from the 3’-end,

ii) AddAB or RecJ, in concert with a RecQ-like helicase (RecQ or RecS), creates 3’-ssDNA tails at the break, and RecN recruits recombination proteins to form a repair centre,

iii) various mediators (e.g., RecO, RecU) modulate the activity of the recombinase RecA,

iv) the RecO mediator mediates second end capture, and

v) the branch migration helicases (RecG and RuvAB), and the resolvase (RecU) process recombination intermediates.

In the second model system, we showed that

i) the role of pSM19035 global regulator ω protein (a dimer in solution, ω₂) is to control the fine-tuning of plasmid copy-number, plasmid segregation, and ε and ζ expression. In addition,

ii) the partitioning protein ω₂ binds to a centromeric-like region, forming a left-handed protein matrix surrounding the straight parS DNA (partition complex, PC), and protein δ (a dimer in solution, δ₂) hydrolyses ATP preferentially in the presence of ω₂ bound to parS (segrosome complex, SC) or non-parS DNA (dynamic complex, DC) and binds DNA after interacting with ω₂, promoting plasmid pairing (bridging complex, BC) at parS DNA or pseudo bridging complex (“BC”) at non-parS DNA. Finally,

iii) the δ₂ disassembly from DNA is dependent on the stoichiometry of the ω₂:δ₂ complex.