The helicase DinG responds to stress due to DNA double strand breaks

Abstract

Neisseria meningitidis (Nm) is a Gram-negative nasopharyngeal commensal that can cause septicaemia and meningitis. The neisserial DNA damage-inducible protein DinG is a helicase related to the mammalian helicases XPD and FANCJ. These helicases belong to superfamily 2, are ATP dependent and exert 5′ → 3′ directionality. To better understand the role of DinG in neisserial genome maintenance, the Nm DinG (DinGNm) enzymatic activities were assessed in vitro and phenotypical characterization of a dinG null mutant (NmΔdinG) was performed. Like its homologues, DinGNm possesses 5′ → 3′ directionality and prefers DNA substrates containing a 5′-overhang. ATPase activity of DinGNm is strictly DNA-dependent and DNA unwinding activity requires nucleoside triphosphate and divalent metal cations. DinGNm directly binds SSBNm with a Kd of 313 nM. Genotoxic stress analysis demonstrated that NmΔdinG was more sensitive to double-strand DNA breaks (DSB) induced by mitomycin C (MMC) than the Nm wildtype, defining the role of neisserial DinG in DSB repair. Notably, when NmΔdinG cells grown under MMC stress assessed by quantitative mass spectrometry, 134 proteins were shown to be differentially abundant (DA) compared to unstressed NmΔdinG cells. Among the DNA replication, repair and recombination proteins affected, polymerase III subunits and recombinational repair proteins RuvA, RuvB, RecB and RecD were significantly down regulated while TopA and SSB were upregulated under stress condition. Most of the other DA proteins detected are involved in metabolic functions. The present study shows that the helicase DinG is probably involved in regulating metabolic pathways as well as in genome maintenance.

Type IV CRISPR-Cas systems are highly diverse and involved in competition between plasmids

“…Moreover, type IV-mediated gene silencing could serve purposes beyond plasmid-plasmid warfare, such as altering host expression profiles to enhance plasmid propagation and/or stabilise maintenance, all piracy practices which plasmids are known to invoke via diverse mechanisms 59,60,61 . In the context of CRISPRi functionality, for which R-loop formation between the crRNA and the DNA target is key, the common association of DinG with type IV loci appears paradoxical, as it is well documented that the substrate for this helicase are R-loops that block replication fork advancement 62,63,64 . Thus, it is tentative to speculate about the potential regulatory or antagonistic role of the helicase component in the removal of type IV crRNA-DNA hybrids, although the purpose of such a function remains unclear.…”

Serum proteases prevent bacterial biofilm formation: role of kallikrein and plasmin

“…Fractions recovered from AEC were prepared for mass spectrometry analysis. Fractions 8, 13, 16, and 24 were subjected to in-solution tryptic digestion, and the purified peptides were analyzed by mass spectrometry using a data-dependent top-10 method as described in Frye et al [ 36 ]. For peptide separation, a 146-min gradient was used with solvent A {0.1% FA/3% ACN [FA:LC-MS grade (Fluka); ACN: LC-MS grade (Merck)]} and solvent B (0.1% FA/97% ACN) using the following steps: (i) 2% solvent B from start to 5 min, (ii) 2% to 20% solvent B from 5 to 105 min, (iii) 20% to 32% solvent B from 105 to 125 min, (iv) 32% to 95% solvent B from 125 to 126 min and (v) 95% solvent B until 146 min.…”

The Host-Pathogen Interactions and Epicellular Lifestyle of Neisseria meningitidis

“…DNA repair by these enzymes is backed up by a redundant network of enzymes, including the bifunctional DNA glycosylase/glycolyases Nth and MutM, making the meningococcus robust to DNA damage by ROS (Nagorska et al, 2012). The DinG helicase has also been shown to increase survival under oxidative stress due to its role in double-stranded break repair (Frye et al, 2017). Repair of damaged proteins occurs via several pathways.…”