In eukaryotes, the spatial and temporal organization of genome duplication gives rise to distinctive profiles of replication origin usage along the chromosomes. While it has become increasingly clear that these programs are important for cellular physiology, the mechanisms by which they are determined and modulated remain elusive. Replication initiation requires the function of cyclin-dependent kinases (CDKs), which associate with various cyclin partners to drive cell proliferation. Surprisingly ...[more]
Project description:Identification of sites of replication initiation by copy number analysis, comparing samples before and after entry into S phase. Experiments were performed in the following strains and conditions: cdc25-22 (temperature-sensitive mutation arrests cells at G2/M for synchrony, otherwise wild type), G2 arrest; Cdc13-Cdc2, G2 arrest. Overall design: Comparison of samples before and after S phase entry; dye-swap experiments were performed and averaged.
Project description:ChIP-on-chip of Cdc45 in synchronized cells operating with the analog-sensitive CDK fusion protein (Cdc13-Cdc2). Cells were released from a G2 block or from a G2 block followed by an extended G1 (G1+ 15 min extension). For the G2 block, samples were collected 30 minutes after release. For the G1+15 extension, samples were collected 3 minutes after release. Timepoint of collection was based on qPCR experiments showing Cdc45 binding at origins of replication. Overall design: Each IP was compared to its reciprocially labeled input sample. Duplicates for every condition