Near-continuously synthesized leading strands in Escherichia coli are broken by ribonucleotide excision
| Autor: | Andrei Kuzminov, Elena A. Kouzminova, Glen E. Cronan |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
chemistry.chemical_classification
0303 health sciences DNA ligase Multidisciplinary Ribonucleotide Okazaki fragments 030306 microbiology Ribonucleotide excision repair DNA replication medicine.disease_cause Cell biology 03 medical and health sciences chemistry medicine Replisome Escherichia coli 030304 developmental biology Nucleotide excision repair |
| Zdroj: | Proceedings of the National Academy of Sciences. 116:1251-1260 |
| ISSN: | 1091-6490 0027-8424 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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