FORK-seq: replication landscape of the Saccharomyces cerevisiae genome by nanopore sequencing.

Genome replication mapping methods profile cell populations, masking cell-to-cell heterogeneity. Here, we describe FORK-seq, a nanopore sequencing method to map replication of single DNA molecules at 200-nucleotide resolution. By quantifying BrdU incorporation along pulse-chased replication intermediates from Saccharomyces cerevisiae, we orient 58,651 replication tracks reproducing population-based replication directionality profiles and map 4964 and 4485 individual initiation and termination events, respectively. Although most events cluster at known origins and fork merging zones, 9% and 18% of initiation and termination events, respectively, occur at many locations previously missed. Thus, FORK-seq reveals the full extent of cell-to-cell heterogeneity in DNA replication.

Using DNA damage sensitivity phenotypes to characterize mutations affecting proteasome function.

Most mutants affected either in the proteasome biogenesis or function accumulate polyubiquitylated proteins and display growth defects at 37°C or in the presence of canavanine, an arginine analog that impairs protein synthesis. We uncovered a new striking phenotype related to DNA damage for some proteasome mutants: mutant strains grew better than the wild type in the presence of specific genotoxic agents (4NQO, Cpt, and MMS). Hyperresistance to 4NQO or Cpt is a new sensitive tool to detect proteasomal defects. Here, we describe simple methods that can be used to show and quantitatively measure this phenotype in budding yeast.