Deinococcus radiodurans, the model extremophile for extreme radiation resistance, encodes two LexA paralogs (LexA1/DR_A0344 and LexA2/DR_0074). Both are cleaved in a RecA-dependent manner, yet neither controls recA or DNA-repair gene induction, whose mechanism (the IrrE/PprI metalloprotease that cleaves the repressor DdrO) was discovered in 2014-2024. What remains open from the 2007 lexA1 lexA2 double-mutant study is the regulatory function of the two LexA proteins themselves: their direct in vivo DNA targets and binding specificity are unknown, and the mechanistic basis of the double mutant's phenotype - transcriptional up-regulation of antioxidant and repair genes, slower growth, and increased resistance to UV and hydrogen peroxide - has never been explained. This open core is the problem addressed here.

Background

Deinococcus radiodurans is a bacterium famous for surviving doses of ionizing radiation that shatter its genome into hundreds of fragments, repairing them accurately. In most bacteria, DNA damage triggers the SOS response: the RecA protein (a DNA recombinase that polymerizes on single-stranded DNA generated at damaged replication forks) stimulates self-cleavage of the LexA repressor, which normally sits on specific operator sequences (LexA boxes) blocking transcription of DNA repair genes; cleavage of LexA lifts this repression. D. radiodurans broke this paradigm. Its genome encodes two LexA paralogs (paralogous genes related by duplication within a genome), LexA1 (DR_A0344) and LexA2 (DR_0074), and both proteins can undergo RecA-dependent cleavage in vitro and in vivo, yet genetic studies showed that neither is involved in inducing recA or the other DNA repair genes after irradiation (Narumi et al. 2001; Sheng et al. 2004; Satoh et al. 2006). Zhou et al. (2007) constructed the lexA1 lexA2 double mutant: it grew more slowly, was more resistant to UV and hydrogen peroxide than wild type, still did not constitutively overexpress RecA, and showed broad transcriptional changes affecting antioxidant, repair, cell-division, protein-synthesis, and transcription-regulation genes. They concluded that a new, LexA-independent mechanism must control the damage-induced proteins. That new mechanism was subsequently found: a two-protein radiation/desiccation response (RDR) system. The XRE-family transcriptional repressor DdrO (XRE denotes a large family of helix-turn-helix bacterial regulators) binds a conserved 17-bp palindromic motif (the RDR motif) upstream of recA and roughly twenty-plus other stress genes; upon DNA damage, the zinc-dependent metalloprotease IrrE, also called PprI, cleaves DdrO, inactivating it and lifting repression. Genome-wide ChIP-seq (chromatin immunoprecipitation coupled to sequencing, an assay that maps where a protein binds DNA in vivo) and RNA-seq defined the DdrO regulon (Eugénie et al. 2021); structural work explained the derepression mechanism; and the upstream activating signal was identified as single-stranded DNA sensed directly by PprI (Lu et al. 2024), with redox signaling through zinc also contributing (Magerand et al. 2021). With the induction mechanism solved, one piece of the original puzzle conspicuously remains: what do LexA1 and LexA2 actually do? LexA2 overexpression down-regulates radioresistance (Satoh et al. 2006), so the paralogs have real physiological functions; putative LexA-binding sites have been mapped only computationally (Khan et al. 2008); and dedicated reviews in 2021 (Blanchard and de Groot) and 2024 (Misra and Rajpurohit) state that the genes regulated by the two D. radiodurans LexA proteins are still unknown. Because both paralogs are RecA-cleavable, their targets, if any, would define a damage-modulated regulon distinct from both the classical SOS system and the IrrE/PprI-DdrO system.

Problem Statement

Define the regulatory function of the LexA1 (DR_A0344) and LexA2 (DR_0074) proteins of Deinococcus radiodurans R1, the surviving open core of the question raised by the lexA1 lexA2 double-mutant analysis of Zhou et al. (2007). Specifically: (1) identify the direct in vivo DNA targets of LexA1 and of LexA2, genome-wide and separately attributed to each paralog, together with the binding specificity (binding motif and operator architecture) they recognize, under basal growth and under relevant stress conditions (e.g., recovery after ionizing radiation and oxidative/H2O2 challenge, states in which RecA-dependent cleavage of these proteins occurs); (2) determine which of the transcriptional changes observed in the lexA1 lexA2 double mutant - up-regulation of antioxidant and repair genes, and altered expression of cell-division, protein-synthesis, and transcription-regulation genes - are direct consequences of losing LexA1/LexA2 repression and which are indirect (for example, cross-talk with the IrrE/PprI-DdrO radiation/desiccation response regulon or with other stress regulators such as DdrI); and (3) explain how the direct LexA-dependent effects account for the double mutant's organismal phenotype of slower growth with increased resistance to UV and hydrogen peroxide. The already-resolved part of the source question - the LexA-independent mechanism that induces recA and repair genes (the IrrE/PprI-DdrO system) - is not part of this problem. If rigorous assays establish that a paralog's direct-target set is empty under the tested conditions, goals (2) and (3) still require a mechanistic attribution (for example, via indirect routes such as cross-talk with the IrrE/PprI-DdrO or DdrI regulons) of the documented double-mutant transcriptional changes and phenotype.

References

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