DrRecQ regulates guanine quadruplex DNA structure dynamics and its impact on radioresistance in Deinococcus radiodurans

Abstract

Summary The GC‐rich genome of Deinococcus radiodurans contains a very high density of putative guanine quadruplex (G4) DNA motifs and its RecQ (drRecQ) was earlier characterized as a 3′→5′ dsDNA helicase. We saw that N‐Methyl mesoporphyrin IX (NMM), a G4 DNA binding drug affected normal growth as well as the gamma radiation resistance of the wild‐type bacterium. Interestingly, NMM treatment and recQ deletion showed additive effect on normal growth but there was no effect of NMM on gamma radiation resistance of recQ mutant. The recombinant drRecQ showed ~400 times higher affinity to G4 DNA (Kd = 11.74 ± 1.77 nM) as compared to dsDNA (Kd = 4.88 ± 1.30 µM). drRecQ showed ATP independent helicase function on G4 DNA, which was higher than ATP‐dependent helicase activity on dsDNA. Unlike wild‐type cells that sparingly stained for G4 structure with Thioflavin T (ThT), recQ mutant showed very high‐density of ThT fluorescence foci on DNA indicating an important role of drRecQ in regulation of G4 DNA structure dynamics in vivo. These results together suggested that drRecQ is an ATP independent G4 DNA helicase that plays an important role in the regulation of G4 DNA structure dynamics and its impact on radioresistance in D. radiodurans.

PprA interacts with replication proteins and affects their physicochemical properties required for replication initiation inDeinococcus radiodurans

“…The unwinding of double strand DNA substrates (with 5' overhang as well as 3' overhang) 448 by drDnaB (2µM) in the presence of different combinations of 1 mM ATP and 2 µM PprA 449 was monitored using fluorescent resonance energy transfer (FRET) approach using 96-well 450 plates as described in (Khairnar et al, 2019). In brief, the substrates used were FAM and 451 BHQ labelled complementary oligonucleotides (Table S1).…”

“…The 459 fluorescence signals were monitored at an interval of 30 seconds using microplate reader 460 (Biotek Synergy H1). 461Similarly, gel-based DNA helicase activity assay of drDnaB was carried out as descried in 462(Khairnar et al, 2019). In brief, the 3′ overhang substrate was made by annealing the [ 32 PDas and Misra, 2012).…”

Genomic Instability of G-Quadruplex Sequences in Escherichia coli: Roles of DinG, RecG, and RecQ Helicases

“…RecQ belongs to a conserved family of helicases important for genome maintenance [ 30 ] (see above, WRM and BLM). The RecQ family of helicases can unwind G-quadruplex structures [ 31 , 32 ]. Nevertheless, while in vitro approaches have shown that bacterial RecQ can resolve G4s, it is unclear whether it plays a role in vivo [ 27 ].…”