The Mycobacterial LexA/RecA-Independent DNA Damage Response Is Controlled by PafBC and the Pup-Proteasome System

Abstract

Mycobacteria exhibit two DNA damage response pathways: the LexA/RecA-dependent SOS response and a LexA/RecA-independent pathway. Using a combination of transcriptomics and genome-wide binding site analysis, we demonstrate that PafBC (proteasome accessory factor B and C), encoded in the Pup-proteasome system (PPS) gene locus, is the transcriptional regulator of the predominant LexA/RecA-independent pathway. Comparison of the resulting PafBC regulon with the DNA damage response of Mycobacterium smegmatis reveals that the majority of induced DNA repair genes are upregulated by PafBC. We further demonstrate that RecA, a member of the PafBC regulon and principal regulator of the SOS response, is degraded by the PPS when DNA damage stress has been overcome. Our results suggest a model for the regulation of the mycobacterial DNA damage response that employs the concerted action of PafBC as master transcriptional activator and the PPS for removal of DNA repair proteins to maintain a temporally controlled stress response.

Preexisting variation in DNA damage response predicts the fate of single mycobacteria under stress

“…we severely impair the frequency and magnitude of RecA pulses, in agreement with the presence of a second possible positive regulator, recently identified in PafBC (Müller et al, 2018). Bars shaded in plum indicate moderately pulsing cells (n = 185), and magenta bars indicate highly pulsing cells (n = 57).…”

“…By deconstructing the recA regulatory region, we show that functional LexA binding is required for pulses to occur and for induction by alkylating agents. Moreover, we observe that ClgR exerts a positive regulation of recA basal expression, consistent with bulk-cell studies (Wang et al, 2011), and with the induction of Clp proteases (Estorninho et we severely impair the frequency and magnitude of RecA pulses, in agreement with the presence of a second possible positive regulator, recently identified in PafBC (Müller et al, 2018). PafBC binding to the recA P1 promoter region was found to be inconclusive and less specific than ClgR binding (Wang et al, 2011;Fudrini Olivencia et al, 2017).…”

“…The data shown are from three independent experiments. We seldom detect dynamic bundles and intense fluorescence, and only in cells that will die, implying that undue amounts of RecA are harmful, and that RecA degradation (Müller et al, 2018) must be fine-tuned in line with transcription toward DNA damage resolution. E, F Single-cell size (E) and growth rate (F) averaged over the lifetime of the cell (58 < n < 159).…”

“…While in model organisms the regulation of the SOS response is based on a single-input inhibitory module that is derepressed by ssDNA (Alon, 2007), the recA regulatory network in mycobacteria is more complex and yet enigmatic (Gopaul et al, 2003;Gamulin et al, 2004). RecA binding to ssDNA elicits LexA self-cleavage and activates the distal promoter (Smollett et al, 2012), whereas transcriptional activators allegedly act on the proximal promoter (Wang et al, 2011;Müller et al, 2018). Lastly, RecX inhibits the nucleoprotein filament formation to shutdown the process (Le et al, 2014).…”

“…Previously, it was shown that recA is dually regulated by both a distal promoter (P2), containing the consensus Cheo box for the SOS-repressor LexA , and by a proximal promoter (P1), containing a pseudo-palindromic sequence that is bound in vitro by the positive regulator ClgR, and whose deletion impairs RecA levels (Gopaul et al, 2003;Russo et al, 2009;Wang et al, 2011). Recently, a second regulator has also been proposed as an alternate P1 activator (Müller et al, 2018). Induction of recA is further influenced by LexA self-cleavage, which is presumably induced by nucleoprotein filaments formed by RecA on damaged ssDNA.…”