Oxidative DNA damage is instrumental in hyperreplication stress-induced inviability of Escherichia coli

Abstract

In Escherichia coli, an increase in the ATP bound form of the DnaA initiator protein results in hyperinitiation and inviability. Here, we show that such replication stress is tolerated during anaerobic growth. In hyperinitiating cells, a shift from anaerobic to aerobic growth resulted in appearance of fragmented chromosomes and a decrease in terminus concentration, leading to a dramatic increase in ori/ter ratio and cessation of cell growth. Aerobic viability was restored by reducing the level of reactive oxygen species (ROS) or by deleting mutM (Fpg glycosylase). The double-strand breaks observed in hyperinitiating cells therefore results from replication forks encountering single-stranded DNA lesions generated while removing oxidized bases, primarily 8-oxoG, from the DNA. We conclude that there is a delicate balance between chromosome replication and ROS inflicted DNA damage so the number of replication forks can only increase when ROS formation is reduced or when the pertinent repair is compromised.

Bactericidal Antibiotics Induce Toxic Metabolic Perturbations that Lead to Cellular Damage

“…For example, recent work has provided evidence that even the very low endogenous levels of 8-oxo-dG in DNA can prove lethal if cells hyper-initiate DNA replication (Charbon et al, 2014). Also, simply increasing the levels of DinB (DNA pol IV) can prove lethal due to the increased incorporation of 8-oxo-dG into DNA (Dwyer et al, 2014; Foti et al, 2012).…”

“…Closely spaced lesions, including tandem lesions containing 8-oxo-dG that are generated by the reaction of pyrimidine peroxyl radicals (Bergeron et al, 2010), can interfere with the process of base excision repair (Bergeron et al, 2010; Cunniffe et al, 2014; Eccles et al, 2010) and could block replication. Alternatively, incomplete base excision repair intermediates could stall DNA polymerase and lead to a lethal collision between two replication forks or stall RNA polymerase leading to a collision between a replication fork and stalled transcription apparatus (Charbon et al, 2014; Merrikh et al, 2012). Also, as 8-oxo-guanine has a lower redox potential than any of the four normal bases, it can be further oxidized to more toxic products, including spiroiminodihydantoin and guanidinohydantoin (Neeley and Essigmann, 2006).…”

Lethality of MalE-LacZ hybrid protein shares mechanistic attributes with oxidative component of antibiotic lethality

“…With respect to tolerating 8-oxo-dG, aerobically grown E. coli live so close to the edge that decreasing the level of MutT by only a factor of two increases the mutation rate (44). Moreover, for aerobically grown E. coli, simply increasing the frequency of initiation of DNA replication (45) or increasing DNA polymerase IV, which has a propensity to use 8-oxo-dGTP (14,18), is enough to cause cell death because BER cannot be completed before encountering the next replication fork (45). An advantage that bacteria gain by living so close to this threshold is that they can mutate in response to stress by increasing the incorporation of oxidized deoxynucleotides into DNA while simultaneously suppressing mismatch repair (30).…”

“…This DNA-based death mechanism, which initially can be counteracted by RecA-dependent or RecB/RecF-dependent processes, is likely complex. Since it takes only a single unrepaired double-stand break (DSB) to kill a bacterial cell (47), one problem could be DSBs caused by MutM and MutY incisions at closely spaced lesions (14) or by replication forks encountering unrepaired BER intermediates (45). Other potentially lethal DNA problems include interstrand crosslinks mediated by abasic sites (48), LigA/MutY-dependent futile cycles of ligation/incision (49), and unresolvable collisions caused by stalled transcription or replicative complexes (50).…”

A combinatorial role for MutY and Fpg DNA glycosylases in mutation avoidance in Mycobacterium smegmatis

“…In mycobacteria, the absence of MutY under oxidative stress conditions could result in an increase in the prevalence of OCDLs, which will impair DNA synthesis -leading to cell death. Furthermore, during the repair of 8-oxo-Gua, double stranded DNA breaks (DSB) can occur if these lesions are closely spaced or encountered by a replication fork and frequent encounters of such events could overwhelm the DNA repair machinery and result in cell death [61]. In B. subtilis, MutY is hypothesized to compete for mispaired bases, which are otherwise repaired by MMR and consistent with this, a carefully coordinated equilibrium is maintained between MutY and MMR [62].…”