Stalled replication fork repair and misrepair during thymineless death in Escherichia coli.
Starvation for DNA precursor dTTP, known as 'thymineless death' (TLD), kills bacterial and eukaryotic cells alike. Despite numerous investigations, toxic mechanisms behind TLD remain unknown, although wrong nucleotide incorporation with subsequent excision dominates the explanations. We show that kinetics of TLD in Escherichia coli is not affected by mutations in DNA repair, ruling out excision after massive misincorporation as the cause of TLD. We found that the rate of DNA synthesis in thymine-starved cells decreases exponentially, indicating replication fork stalling. Processing of stalled replication forks by recombinational repair is known to fragment the chromosome, and we detect significant chromosomal fragmentation during TLD. Moreover, we report that, out of major recombinational repair functions, only inactivation of recF and recO relieves TLD, identifying the poisoning mechanism. Inactivation of recJ and rep has slight effect, while the recA, recBC, ruvABC, recG and uvrD mutations all accelerate TLD, identifying the protection mechanisms. Our epistatic analysis argues for two distinct pathways protecting against TLD: RecABCD/Ruv repairs the double-strand breaks, whereas UvrD counteracts RecAFO-catalyzed toxic single-strand gap processing.
Kuong
Kawai J
KJ
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Kuzminov
Andrei
A
D002876:Chromosomes, Bacterial / Q000235:genetics; D053938:DNA Fragmentation; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D050296:Microbial Viability / Q000235:genetics; D008957:Models, Genetic; D051719:MutS DNA Mismatch-Binding Protein / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D013014:SOS Response, Genetics / Q000235:genetics; D013941:Thymine / Q000378:metabolism*
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C081006:RecO protein, E coli; C489340:recF protein, E coli; D011938:Rec A Recombinases; C506746:UvrD protein, E coli; D051719:MutS DNA Mismatch-Binding Protein; D004265:DNA Helicases; D013941:Thymine
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