Detection and Sequencing of Okazaki Fragments in S. cerevisiae.
We have previously demonstrated that lagging-strand synthesis in budding yeast is coupled with chromatin assembly on newly synthesized DNA. Using a strain of S. cerevisiae in which DNA ligase I can be conditionally depleted, we can enrich and purify Okazaki fragments. We delineate a method to extract, end label, and visualize Okazaki fragments using denaturing agarose gel electrophoresis. Furthermore, we describe an ion-exchange chromatographic method for purification of fragments and preparation of strand-specific sequencing libraries. Deep sequencing of Okazaki fragments generates a comprehensive, genomic map of DNA synthesis, starting from a single asynchronous culture. Altogether this approach represents a tractable system to investigate key aspects of DNA replication and chromatin assembly.
Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY, 10065, USA, duncan.smith@nyu.edu.
Duncan J
DJ
Smith
Tejas
T
Yadav
Iestyn
I
Whitehouse
D004247:DNA / Q000235:genetics*; D004271:DNA, Fungal / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D016681:Genome, Fungal; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013194:Staining and Labeling
D004271:DNA, Fungal; C022262:Okazaki fragments; D004247:DNA