Post-replicative nick translation occurs on the lagging strand during prolonged depletion of DNA ligase I in Saccharomyces cerevisiae

Natasha C. Koussa, Duncan J. Smith

Research output: Contribution to journalArticlepeer-review

Abstract

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 50 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.

Original languageEnglish (US)
Article numberjkab205
JournalG3: Genes, Genomes, Genetics
Volume11
Issue number8
DOIs
StatePublished - Aug 2021

Keywords

  • DNA ligase I
  • DNA polymerase delta
  • DNA replication
  • Lagging-strand synthesis

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Genetics(clinical)

Fingerprint

Dive into the research topics of 'Post-replicative nick translation occurs on the lagging strand during prolonged depletion of DNA ligase I in Saccharomyces cerevisiae'. Together they form a unique fingerprint.

Cite this