Phosphorylation of Deinococcus radiodurans RecA Regulates Its Activity and May Contribute to Radioresistance

Abstract

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.Deinococcus radiodurans has a remarkable capacity to survive extreme doses of radiations and other DNA-damaging agents. Studies aimed at unraveling the molecular bases for these unusual properties have revealed that D. radiodurans encodes mechanisms for highly efficient DNA double strand break (DSB) 2 repair and oxidative stress management (1-3). DSB repair in this Gram-positive bacterium is accomplished in two phases during post-irradiation recovery (PIR); phase I is dominated by extended synthesis-dependent strand annealing (ESDSA) processes, whereas phase II involves slow crossover events in homologous recombination leading to the repair and re-establishment of the multipartite D. radiodurans genome structure (4). Despite the fact that the two phases of PIR have DNA substrates of different structures and topologies, D. radiodurans RecA (DrRecA) is required throughout DSB repair during PIR (5). Biochemical characterization of recombinant DrRecA revealed that it can form a filament on singlestranded DNA (ssDNA), exhibit co-protease activity, and utilize ATP or dATP for its energy requirements, akin to other bacterial RecA proteins (6), but it also has unusual properties. In contrast to most bacterial RecA proteins, DrRecA promotes inverse strand exchange reactions (7). Also, DrRecA promotes DNA degradation during the early phase of ESDSA repair (5), which is opposite to the function observed with Escherichia coli RecA. Transcription of DrRecA is induced in response to ␥ radiation (8, 9). However, the mechanisms by which ␥ radiation induces DrRecA expression are unusual. Inactivation of both D. radiodurans lexA genes does not attenuate ␥ radiation induction of DrRecA expression (10, 11). Thus, in contrast to many bacteria, LexA...

Guanine Quadruplex DNA Regulates Gamma Radiation Response of Genome Functions in the Radioresistant Bacterium Deinococcus radiodurans

“…For instance, DdrO (DR_2574) has been identified as a repressor of gene expression, which upon cleavage by PprI (IrrE) depresses the expression of DNA damage response genes (39). Regulation of RecA activity by phosphorylation has been demonstrated recently (40). It has been shown that RecA and PprA of D. radiodurans are phosphorylated by a DNA damage response protein kinase and that the phosphorylation of these proteins affects their functions both in vivo and in vitro (40,41).…”

“…Regulation of RecA activity by phosphorylation has been demonstrated recently (40). It has been shown that RecA and PprA of D. radiodurans are phosphorylated by a DNA damage response protein kinase and that the phosphorylation of these proteins affects their functions both in vivo and in vitro (40,41). These mechanisms, however, could explain the gamma radiationinducible expression of only some of the genes in the absence of a canonical SOS response.…”

Tyrosine Phosphorylation as a Widespread Regulatory Mechanism in Prokaryotes

“…It is likely that SSB is dephosphorylated during the DNA damage response, modulating its binding to DNA and allowing RecA-dependent DNA repair to occur. In another paradigm of DNA damage responses with Deinococcus radiodurans, phosphorylation of RecA at Tyr77 and additionally at Thr318 modifies the activity of RecA by increasing its affinity for double-stranded DNA (dsDNA) (40). It is unclear whether this occurs for RecA homologues in other bacteria, although the above examples of tyrosine phosphorylation of DNA binding proteins involved in DNA repair and recombination mechanisms warrant a fresh look at the dynamic nature of stress responses in bacteria.…”

RPA3 is a potential marker of prognosis and radioresistance for nasopharyngeal carcinoma

“…DSBs are major lesions induced by irradiation , and the DSB repair capacity is closely related to radiosensitivity . Because RPA3 reportedly participates in the HR of DSBs , we evaluated the DNA damage response induced by 2 Gy of irradiation in these cells.…”