title: Chromosome demise in the wake of ligase-deficient replication.
abstract: Bacterial DNA ligases, NAD⁺-dependent enzymes, are distinct from eukaryotic ATP-dependent ligases, representing promising targets for broad-spectrum antimicrobials. Yet, the chromosomal consequences of ligase-deficient DNA replication, during which Okazaki fragments accumulate, are still unclear. Using ligA251(Ts), the strongest ligase mutant of Escherichia coli, we studied ligase-deficient DNA replication by genetic and physical approaches. Here we show that replication without ligase kills after a short resistance period. We found that double-strand break repair via RecA, RecBCD, RuvABC and RecG explains the transient resistance, whereas irreparable chromosomal fragmentation explains subsequent cell death. Remarkably, death is mostly prevented by elimination of linear DNA degradation activity of ExoV, suggesting that non-allelic double-strand breaks behind replication forks precipitate DNA degradation that enlarge them into allelic double-strand gaps. Marker frequency profiling of synchronized replication reveals stalling of ligase-deficient forks with subsequent degradation of the DNA synthesized without ligase. The mechanism that converts unsealed nicks behind replication forks first into repairable double-strand breaks and then into irreparable double-strand gaps may be behind lethality of any DNA damaging treatment.
authors.affiliation: Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801-3709, USA.
authors.forename: Elena A
authors.initials: EA
authors.lastname: Kouzminova
authors.forename: Andrei
authors.initials: A
authors.lastname: Kuzminov
mesh_terms: D002876:Chromosomes, Bacterial / Q000378:metabolism*; D053938:DNA Fragmentation; D011088:DNA Ligases / Q000172:deficiency / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics / Q000254:growth & development* / Q000502:physiology; D029968:Escherichia coli Proteins / Q000378:metabolism*; D043211:Exodeoxyribonuclease V / Q000235:genetics / Q000378:metabolism; D050296:Microbial Viability; D009154:Mutation; D013696:Temperature
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D011088:DNA Ligases; C497118:LigA protein, E coli