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Proc Natl Acad Sci U S A. 2019 Jan 22;116(4):1251-1260. doi: 10.1073/pnas.1814512116. Epub 2019 Jan 7.

Near-continuously synthesized leading strands in Escherichia coli are broken by ribonucleotide excision.

Author information

1
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801.
2
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801 kuzminov@illinois.edu.

Abstract

In vitro, purified replisomes drive model replication forks to synthesize continuous leading strands, even without ligase, supporting the semidiscontinuous model of DNA replication. However, nascent replication intermediates isolated from ligase-deficient Escherichia coli comprise only short (on average 1.2-kb) Okazaki fragments. It was long suspected that cells replicate their chromosomal DNA by the semidiscontinuous mode observed in vitro but that, in vivo, the nascent leading strand was artifactually fragmented postsynthesis by excision repair. Here, using high-resolution separation of pulse-labeled replication intermediates coupled with strand-specific hybridization, we show that excision-proficient E. coli generates leading-strand intermediates >10-fold longer than lagging-strand Okazaki fragments. Inactivation of DNA-repair activities, including ribonucleotide excision, further increased nascent leading-strand size to ∼80 kb, while lagging-strand Okazaki fragments remained unaffected. We conclude that in vivo, repriming occurs ∼70× less frequently on the leading versus lagging strands, and that DNA replication in E. coli is effectively semidiscontinuous.

KEYWORDS:

Okazaki fragments; ligase mutant; replication intermediates; ribonucleotide excision repair; the leading strand

Comment in

PMID:
30617079
PMCID:
PMC6347710
DOI:
10.1073/pnas.1814512116
[Indexed for MEDLINE]
Free PMC Article

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