Post-replicative nick translation occurs on the lagging strand during prolonged depletion of DNA ligase I in Saccharomyces cerevisiae
| Autor: | Duncan J. Smith, Natasha C. Koussa |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
AcademicSubjects/SCI01140
Saccharomyces cerevisiae Proteins DNA polymerase AcademicSubjects/SCI00010 DNA ligase I Saccharomyces cerevisiae DNA replication DNA polymerase delta QH426-470 AcademicSubjects/SCI01180 03 medical and health sciences chemistry.chemical_compound DNA Ligase ATP 0302 clinical medicine Genetics Nick translation Molecular Biology Genetics (clinical) Polymerase 030304 developmental biology DNA Polymerase III chemistry.chemical_classification Investigation 0303 health sciences DNA ligase biology Okazaki fragments Cell biology chemistry biology.protein AcademicSubjects/SCI00960 lagging-strand synthesis 030217 neurology & neurosurgery DNA |
| Zdroj: | G3: Genes, Genomes, Genetics, Vol 11, Iss 8 (2021) G3: Genes|Genomes|Genetics |
| ISSN: | 2160-1836 |
| DOI: | 10.1093/g3journal/jkab205 |
| Popis: | During lagging-strand synthesis, strand-displacement synthesis by DNA polymerase delta (Pol ∂), coupled to nucleolytic cleavage of DNA flap structures, produces a nick-translation reaction that replaces the DNA at the 5′ end of the preceding Okazaki fragment. Previous work following depletion of DNA ligase I in Saccharomyces cerevisae suggests that DNA-bound proteins, principally nucleosomes and the transcription factors Abf1/Rap1/Reb1, pose a barrier to Pol ∂ synthesis and thereby limit the extent of nick translation in vivo. However, the extended ligase depletion required for these experiments could lead to ongoing, non-physiological nick translation. Here, we investigate nick translation by analyzing Okazaki fragments purified after transient nuclear depletion of DNA ligase I in synchronized or asynchronous Saccharomyces cerevisiae cultures. We observe that, even with a short ligase depletion, Okazaki fragment termini are enriched around nucleosomes and Abf1/Reb1/Rap1-binding sites. However, protracted ligase depletion leads to a global change in the location of these termini, moving them toward nucleosome dyads from a more upstream location and further enriching termini at Abf1/Reb1/Rap1-binding sites. In addition, we observe an under-representation of DNA derived from DNA polymerase alpha—the polymerase that initiates Okazaki fragment synthesis—around the sites of Okazaki termini obtained from very brief ligase depletion. Our data suggest that, while nucleosomes and transcription factors do limit strand-displacement synthesis by Pol ∂ in vivo, post-replicative nick translation can occur at unligated Okazaki fragment termini such that previous analyses represent an overestimate of the extent of nick translation occurring during normal lagging-strand synthesis. |
| Databáze: | OpenAIRE |
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