No abstract

DrRecA and PprA proteins function are crucial for the extraordinary resistance to γ-radiation and DNA strand break repair in Deinococcus radiodurans. DrRecA mediated homologous recombination help in DNA strand break repair and cell survival, while the PprA protein confers radio-resistance via its roles in DNA repair, genome maintenance, and cell division. Genetically recA and pprA genes interact and constitute an epistatic group however, the mechanism underlying their functional interaction is not clear. Here, we showed the physical and functional interaction of DrRecA and PprA protein both in solution and inside the cells. The absence of the pprA gene increases the recombination frequency in gamma-irradiated D. radiodurans cells and genomic instability in cells growing under normal conditions. PprA negatively regulates the DrRecA functions by inhibiting DrRecA mediated DNA strand exchange and ATPase function in vitro. Furthermore, it is shown that the inhibitory effect of PprA on DrRecA catalyzed DNA strand exchange was not due to sequestration of homologous dsDNA and was dependent on PprA oligomerization and DNA binding property. Together, results suggest that PprA is a new member of recombination mediator proteins (RMPs), and able to regulate the DrRecA function in γ-irradiated cells by protecting the D. radiodurans genome from hyper-recombination and associated negative effects.

Deinococcus radiodurans has a remarkable capacity to survive exposure to extreme levels of radiation that cause hundreds of DNA double strand breaks (DSBs). DSB repair in this bacterium depends on its recombinase A protein (DrRecA). DrRecA plays a pivotal role in both extended synthesis-dependent strand annealing and slow crossover events of DSB repair during the organism's recovery from DNA damage. The mechanisms that control DrRecA activity during the D. radiodurans response to ␥ radiation exposure are unknown. Here, we show that DrRecA undergoes phosphorylation at Tyr-77 and Thr-318 by a DNA damage-responsive serine threonine/tyrosine protein kinase (RqkA). Phosphorylation modifies the activity of DrRecA in several ways, including increasing its affinity for dsDNA and its preference for dATP over ATP. Strand exchange reactions catalyzed by phosphorylated versus unphosphorylated DrRecA also differ. In silico analysis of DrRecA structure support the idea that phosphorylation can modulate crucial functions of this protein. Collectively, our findings suggest that phosphorylation of DrRecA enables the recombinase to selectively use abundant dsDNA substrate present during post-irradiation recovery for efficient DSB repair, thereby promoting the extraordinary radioresistance of D. radiodurans. * The authors declare that they have no conflicts of interest with the contents of this article. □ S This article contains supplemental Figs. 1-4. 1 Recipient of a Fulbright-Nehru senior research fellowship grant from the United States-India Education Foundation (USIEF) and the J. William Fulbright foreign scholarship Board.