title: Low-molecular-weight DNA replication intermediates in Escherichia coli: mechanism of formation and strand specificity.
abstract: Chromosomal DNA replication intermediates, revealed in ligase-deficient conditions in vivo, are of low molecular weight (LMW) independently of the organism, suggesting discontinuous replication of both the leading and the lagging DNA strands. Yet, in vitro experiments with purified enzymes replicating sigma-structured substrates show continuous synthesis of the leading DNA strand in complete absence of ligase, supporting the textbook model of semi-discontinuous DNA replication. The discrepancy between the in vivo and in vitro results is rationalized by proposing that various excision repair events nick continuously synthesized leading strands after synthesis, producing the observed LMW intermediates. Here, we show that, in an Escherichia coli ligase-deficient strain with all known excision repair pathways inactivated, new DNA is still synthesized discontinuously. Furthermore, hybridization to strand-specific targets demonstrates that the LMW replication intermediates come from both the lagging and the leading strands. These results support the model of discontinuous leading strand synthesis in E. coli.
authors.affiliation: Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
authors.forename: Luciana
authors.initials: L
authors.lastname: Amado
authors.forename: Andrei
authors.initials: A
authors.lastname: Kuzminov
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D011088:DNA Ligases / Q000172:deficiency; D004258:DNA Polymerase III / Q000235:genetics; D004260:DNA Repair; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D043211:Exodeoxyribonuclease V / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D008970:Molecular Weight; D009154:Mutation; D010641:Phenotype; D016914:Ribonuclease H / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; C065639:DNA polymerase A; D004258:DNA Polymerase III; D005090:Exodeoxyribonucleases; C070765:recJ protein, Bacteria; D043211:Exodeoxyribonuclease V; D016914:Ribonuclease H; C078856:ribonuclease HI; D011088:DNA Ligases
keywords: BER; EDTA; HMW; IMW; LMW; MR; NER; Okazaki fragments; base excision repair; ethylenediaminetetraacetic acid; excision repair; high molecular weight; intermediate molecular weight; ligA mutants; low molecular weight; mismatch repair; nucleotide excision repair; pulse labeling; strand-specific hybridization