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

Abstract

Guanine quadruplex (G4) DNA/RNA are secondary structures that regulate the various cellular processes in both eukaryotes and bacteria. Deinococcus radiodurans, a Gram-positive bacterium known for its extraordinary radioresistance, shows a genomewide occurrence of putative G4 DNA-forming motifs in its GC-rich genome. N-Methyl mesoporphyrin (NMM), a G4 DNA structure-stabilizing drug, did not affect bacterial growth under normal conditions but inhibited the postirradiation recovery of gamma-irradiated cells. Transcriptome sequencing analysis of cells treated with both radiation and NMM showed repression of gamma radiation-responsive gene expression, which was observed in the absence of NMM. Notably, this effect of NMM on the expression of housekeeping genes involved in other cellular processes was not observed. Stabilization of G4 DNA structures mapped at the upstream of recA and in the encoding region of DR_2199 had negatively affected promoter activity in vivo, DNA synthesis in vitro and protein translation in Escherichia coli host. These results suggested that G4 DNA plays an important role in DNA damage response and in the regulation of expression of the DNA repair proteins required for radioresistance in D. radiodurans. IMPORTANCE Deinococcus radiodurans can recover from extensive DNA damage caused by many genotoxic agents. It lacks LexA/RecA-mediated canonical SOS response. Therefore, the molecular mechanisms underlying the regulation of DNA damage response would be worth investigating in this bacterium. D. radiodurans genome is GC-rich and contains numerous islands of putative guanine quadruplex (G4) DNA structure-forming motifs. Here, we showed that in vivo stabilization of G4 DNA structures can impair DNA damage response processes in D. radiodurans. Essential cellular processes such as transcription, DNA synthesis, and protein translation, which are also an integral part of the double-strand DNA break repair pathway, are affected by the arrest of G4 DNA structure dynamics. Thus, the role of DNA secondary structures in DNA damage response and radioresistance is demonstrated.

G-Quadruplexes in the Archaea Domain

“…Ding et al hypothesized that stress-resistant bacteria found in the Deinococcales may utilize putative quadruplex sequences (PQS) for gene regulatory purposes. An enrichment in prokaryote PQS has been found in thermophilic organisms [ 33 ] but also in organisms with resistance to other stress factors, such as radiation [ 36 , 37 ]; thus, a direct correlation between temperature and G4 presence is not supported by these findings. In addition, while bacteria in the Deinococcus-Thermus group are the most abundant for PQS, it is striking that the mostly thermophilic and hyperthermophilic bacteria in the Thermotogae phylum have one of the lowest PQS frequencies.…”

Topoisomerase IB interacts with genome segregation proteins and is involved in multipartite genome maintenance inDeinococcus radiodurans

“…322in small plasmid(Mishra et al, 2019). The change in genome copy number has also been 323 reported when the coding sequences of genome segregation proteins were deleted in this324 bacterium (Maurya et al, 2019a; Maurya et al, 2019b).…”

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31Deinococcus radiodurans, an extremophile, resistant to many abiotic stresses including 32 ionizing radiation, has 2 type I topoisomerases (drTopo IA and drTopo IB) and one type II 33 topoisomerase (DNA gyrase). The role of drTopo IB in guanine quadruplex DNA (G4 DNA) 34 metabolism was shown in vitro. Here we report that D. radiodurans cells lacking drTopo IB 35 (∆topoIB) show sensitivity to G4 DNA binding drug (NMM) under normal growth conditions.

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Topoisomerase IB interacts with genome segregation proteins and is involved in multipartite genome maintenance in Deinococcus radiodurans

“…The cell lytic efficiency was determined using Neubauer counting chamber and the genomic DNA integrity was analysed by agarose gel electrophoresis. The genomic DNA was serially diluted and used for determining genomic copy number by quantitative Real-Time PCR as described in (Mishra et al, 2019). For standardization of qPCR two individual genes for each replicon with almost same PCR efficiency were taken for copy number determination as given in (Mishra et al, 2019).…”