Project description:Antimetabolite chemotherapies increase uracil levels in DNA, and thus identification of factors that influence the uracil content in DNA may have implications for understanding uracil-mediated chromosomal instability. We previously showed in the budding yeast Saccharomyces cerevisiae that uracil content in DNA correlates with replication timing, where the earliest and latest replicating regions are depleted in uracil. Here, we manipulated nucleotide biosynthesis enzymes in budding yeast to determine whether the pattern of uracil incorporation could be altered. In strains with high levels of uracil incorporation, deletion of dCMP deaminase (Dcd1) accelerated uracil incorporation at early-firing origins, likely due to rapid dTTP pool depletion. In contrast, increasing the activity of ribonucleotide reductase, which is required for the synthesis of all dNTPs via ribonucleotide diphosphates, lead to dUTP and dTTP pool equilibration and a concomitant increase in uracil content throughout the genome. These data suggest that uracil availability and the dUTP:dTTP ratio are temporally regulated during S phase and govern uracil incorporation into the genome. Therapeutic manipulation of nucleotide biosynthesis in human cells to either increase the dUTP pool or deplete the dTTP pool in early S phase may therefore improve the efficacy of antimetabolite chemotherapies.
Project description:Cdc7 kinase is known to initiate DNA replication, but it is unknown where Cdc7 is found within the genome. We modified the Calling Cards method that uses the Ty5 retrotransposon to investigate Cdc7 binding in the genome. The Ty5 retrotransposon is inserted into the genome by DNA transcription factors or replication factors binding within the genome. We find that Cdc7 inserts Ty5 transposons throughout chromosomes and furthermore creates more Ty5 insertions into regions of DNA that are known to replicate early. Cdc7 does not solely integrate Ty5 at origins or replication, but rather throughout the genome.
Project description:Rad53-mediated regulation of Rrm3 and Pif1 DNA helicases contributes to prevention of aberrant fork transitions under replication stress.
