Abstract
Origins of DNA replication are specified by the ordered recruitment of replication factors in a cell-cycle–dependent manner. The assembly of the pre-replicative complex in G1 and the pre-initiation complex prior to activation in S phase are well characterized; however, the interplay between the assembly of these complexes and the local chromatin environment is less well understood. To investigate the dynamic changes in chromatin organization at and surrounding replication origins, we used micrococcal nuclease (MNase) to generate genome-wide chromatin occupancy profiles of nucleosomes, transcription factors, and replication proteins through consecutive cell cycles in Saccharomyces cerevisiae. During each G1 phase of two consecutive cell cycles, we observed the downstream repositioning of the origin-proximal +1 nucleosome and an increase in protected DNA fragments spanning the ARS consensus sequence (ACS) indicative of pre-RC assembly. We also found that the strongest correlation between chromatin occupancy at the ACS and origin efficiency occurred in early S phase, consistent with the rate-limiting formation of the Cdc45–Mcm2-7–GINS (CMG) complex being a determinant of origin activity. Finally, we observed nucleosome disruption and disorganization emanating from replication origins and traveling with the elongating replication forks across the genome in S phase, likely reflecting the disassembly and assembly of chromatin ahead of and behind the replication fork, respectively. These results provide insights into cell-cycle–regulated chromatin dynamics and how they relate to the regulation of origin activity.
Highlights
Duplication of a cell’s genetic information occurs every cell cycle in S phase
While many studies have carefully examined the kinetics of DNA replication progression through S phase [3,4,5,6,7], few have examined the chromatin dynamics of replication origins as cells progress through consecutive cell cycles
Cells were released from α-factor arrest and samples were collected every 10 min for approximately two complete cell cycles (150 min) (Figure 1A)
Summary
While DNA replication is restricted to S phase, the DNA replication program is established earlier in the cell cycle with the licensing of DNA replication origins in G1 [1]. These licensed origins are activated with an inherent efficiency during S phase [2]. While many studies have carefully examined the kinetics of DNA replication progression through S phase [3,4,5,6,7], few have examined the chromatin dynamics of replication origins as cells progress through consecutive cell cycles. Understanding how chromatin structure and organization change at DNA replication origins as they progress through consecutive unperturbed cell cycles will provide important insights into the chromatin features that modulate origin usage and efficiency
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