Stressed Dna Replication Generates Stressed Dna

Much of our understanding of eukaryotic replication dynamics, origin, and polymerase usage and replication factors has come from studies using the yeast Saccharomyces cerevisiae. From the definition of replication origins using plasmid transformation (1, 2) and two-dimensional gel electrophoresis (3), identification of replication factors using various genetic screens for cell cycle progression (4) and plasmid stability (5, 6) and genome-wide replication patterns using Okazaki fragment sequencing (7, 8) and strand-specific sequencing of incorporated ribonucleotides in DNA (9), we have learned much about DNA replication under normal and disrupted conditions. As many of the replication factors are conserved, the findings have been applicable to more complex genomes. Additionally, genome instability in yeast arising from perturbations to the normal replication program mirror those found in human cells, particularly the genome instability resulting in loss of heterozygosity (LOH), genome rearrangements, and mutagenesis found in tumor cells and other diseases with DNA repair defects (10, 11).\n\nPerturbations in the DNA replication process can result in regions of single-stranded DNA (ssDNA) accumulating near the replication fork (12, 13). A useful tool in the Saccharomyces arsenal of in vivo genetic tricks is expression of the human APOBEC3B protein to mark the location of ssDNA (14⇓–16). This protein is a member of the cytosine deaminase family and mutates cytosine residues on ssDNA to uracil, resulting in C-to-T mutations. APOBEC3B protein does not naturally occur in yeast but can be expressed from a plasmid introduced into yeast strains. The U residues that arise from C deamination are preserved by deleting the UNG1 gene … \n\n[↵][1]1Email: hannah.klein{at}nyulangone.org.\n\n [1]: #xref-corresp-1-1