Mycobacterium smegmatis PafBC is involved in regulation of DNA damage response

Abstract

Two genes, pafB and pafC, are organized in an operon with the Pup-ligase gene pafA, which is part of the Pup-proteasome system (PPS) present in mycobacteria and other actinobacteria. The PPS is crucial for Mycobacterium tuberculosis resistance towards reactive nitrogen intermediates (RNI). However, pafB and pafC apparently play only a minor role in RNI resistance. To characterize their function, we generated a pafBC deletion in Mycobacterium smegmatis (Msm). Proteome analysis of the mutant strain revealed decreased cellular levels of various proteins involved in DNA damage repair, including recombinase A (RecA). In agreement with this finding, Msm ΔpafBC displayed increased sensitivity to DNA damaging agents. In mycobacteria two pathways regulate DNA repair genes: the LexA/ RecA-dependent SOS response and a predominant pathway that controls gene expression via a LexA/ RecA-independent promoter, termed P1. PafB and PafC feature winged helix-turn-helix DNA binding motifs and we demonstrate that together they form a stable heterodimer in vitro, implying a function as a heterodimeric transcriptional regulator. Indeed, P1-driven transcription of recA was decreased in Msm ΔpafBC under standard conditions and induction of recA expression upon DNA damage was strongly impaired. Taken together, our data indicate an important regulatory function of PafBC in the mycobacterial DNA damage response.Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the most successful human pathogens and represents a major global health problem. Mtb features a multitude of mechanisms that allow the phagocytozed bacteria to withstand the host's innate and adaptive immune response and enable them to persist inside the host macrophages 1,2 . After successful immune evasion the pathogen can persist in the human for decades.A hallmark of activated macrophages is the production of reactive oxygen species and nitric oxide, which damage bacterial proteins, lipids and nucleic acids 3,4 . Thus, in order to maintain the integrity of its genome, Mtb relies on robust mechanisms of DNA damage repair. Whereas in most bacteria the genes involved in these processes are regulated as part of the so-called SOS response, mycobacteria were shown to make use of an additional, independent regulatory mechanism 5-8 . In the SOS response pathway the key regulators are LexA and RecA. Under standard growth conditions the repressor LexA prevents transcription of DNA repair genes by binding to a specific sequence within their promoter, the SOS box 9 . RecA senses DNA damage by binding to single-strand DNA that is generated as a result, thereby forming nucleoprotein filaments. This in turn activates RecA to promote auto-catalytic cleavage of the repressor LexA 9 . As a consequence LexA levels initially decrease and transcription of DNA damage repair genes is de-repressed 10,11 . With only few exceptions, Mtb harbours the genes found in Escherichia coli to mediate a fully functional SOS response 12,13 .Early studies demonstrated that the Mtb recA...

Structure and functional implications of WYL-domain-containing transcription factor PafBC involved in the mycobacterial DNA damage response

“…Subsequently, the dye resazurin was added, which is reduced by living (but not by dead) cells to resorufin, giving rise to a color change. Wild type M. smegmatis cells grow in presence of up to 100 ng/ml MMC, while the ΔpafBC strain shows growth only up to about 8 ng/ml MMC, which is in agreement with the previously determined minimal inhibitory concentrations for these strains (Figure 5a) (Fudrini Olivencia et al, 2017).…”

“…The asymmetric arrangement of PafBC is not surprising, since PafBC is not in complex with its consensus DNA binding site and its domain arrangement reflects the non-activated form of PafBC. This is in agreement with the observation that specific interaction between PafBC and the identified DNA-target regions takes place in vivo, but could not be reconstituted in vitro (Fudrini Olivencia et al, 2017;Müller et al, 2018), further supporting the notion that PafBC is in a non-activated state in absence of the putative DNA-stress sensing ligand.…”

“…Previously, we showed that PafBC levels do not change under stress conditions and we could not detect any specific DNA binding activity towards the RecA-NDp motif in vitro (Fudrini Olivencia et al, 2017;Müller et al, 2018), suggesting that PafBC requires a co-activator for its activity, which is only present during stress conditions. The structural homology between the WYL domains of PafBC and Sm-folds involved in RNA binding indicates that the response-producing ligand might be an RNA molecule and the WYL domain could act as a ligand-sensing domain.…”

“…This predominant pathway operates independently of LexA and RecA, as demonstrated by deletion of recA in Mtb, which leaves upregulation of most DNA repair genes intact (Davis et al, 2002b;Rand et al, 2003). Different from the regulatory principle of derepression, these genes are regulated by transcriptional activation by the heterodimeric protein complex PafBC (Fudrini Olivencia et al, 2017;Müller et al, 2018). The complex consists of the close sequence homologs PafB and PafC (proteasome accessory factors B and C) that are encoded together in an operon that is tightly associated with the bacterial proteasome gene locus, suggesting a functional connection.…”

“…PafBC activates its target genes via a promoter motif called RecA-NDp (RecA-independent promoter), which was demonstrated by in vivo identification of PafBC binding sites using cell culture cross-linking followed by immunoprecipitation of PafBC-DNA complexes (Müller et al, 2018). However, PafBC protein levels are not changing in response to DNA stress (Fudrini Olivencia et al, 2017). Furthermore, specific interaction between PafBC and the identified DNA-target regions could not be reconstituted in vitro.…”