title: The replication intermediates in Escherichia coli are not the product of DNA processing or uracil excision.
abstract: The current model of DNA replication in Escherichia coli postulates continuous synthesis of the leading strand, based on in vitro experiments with purified enzymes. In contrast, in vivo experiments in E. coli and its bacteriophages, in which maturation of replication intermediates was blocked, report discontinuous DNA synthesis of both the lagging and the leading strands. To address this discrepancy, we analyzed nascent DNA species from ThyA+ E. coli cells replicating their DNA in ligase-deficient conditions to block maturation of replication intermediates. We report here that the bulk of the newly synthesized DNA isolated from ligase-deficient cells have a length between 0.3 and 3 kb, with a minor fraction being longer that 11 kb but shorter than the chromosome. The low molecular weight of the replication intermediates is unchanged by blocking linear DNA processing with a recBCD mutation or by blocking uracil excision with an ung mutation. These results are consistent with the previously proposed discontinuous replication of the leading strand in E. coli.
authors.affiliation: Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801-3709, USA.
authors.forename: Luciana
authors.initials: L
authors.lastname: Amado
authors.forename: Andrei
authors.initials: A
authors.lastname: Kuzminov
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000235:genetics; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000378:metabolism; D043211:Exodeoxyribonuclease V / Q000378:metabolism; D009154:Mutation; D009693:Nucleic Acid Hybridization; D051738:Origin Recognition Complex; D014498:Uracil / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; C022262:Okazaki fragments; D051738:Origin Recognition Complex; D014498:Uracil; D004247:DNA; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli; D011088:DNA Ligases