Low-molecular-weight DNA replication intermediates in escherichia coli: Mechanism of formation and strand specificity

Luciana Amado, Andrei Kuzminov

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)4177-4191
Number of pages15
JournalJournal of Molecular Biology
Volume425
Issue number22
DOIs
StatePublished - Nov 15 2013

Keywords

  • Okazaki fragments
  • excision repair
  • ligA mutants
  • pulse labeling
  • strand-specific hybridization

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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