Abstract
BackgroundDuring S. pombe S-phase, initiation of DNA replication occurs at multiple sites (origins) that are enriched with AT-rich sequences, at various times. Current studies of genome-wide DNA replication profiles have focused on the DNA replication timing and origin location. However, the replication and/or firing efficiency of the individual origins on the genomic scale remain unclear.Methodology/Principal FindingsUsing the genome-wide ORF-specific DNA microarray analysis, we show that in S. pombe, individual origins fire with varying efficiencies and at different times during S-phase. The increase in DNA copy number plotted as a function of time is approximated to the near-sigmoidal model, when considering the replication start and end timings at individual loci in cells released from HU-arrest. Replication efficiencies differ from origin to origin, depending on the origin's firing efficiency. We have found that DNA replication is inefficient early in S-phase, due to inefficient firing at origins. Efficient replication occurs later, attributed to efficient but late-firing origins. Furthermore, profiles of replication timing in cds1Δ cells are abnormal, due to the failure in resuming replication at the collapsed forks. The majority of the inefficient origins, but not the efficient ones, are found to fire in cds1Δ cells after HU removal, owing to the firing at the remaining unused (inefficient) origins during HU treatment.Conclusions/SignificanceTaken together, our results indicate that efficient DNA replication/firing occurs late in S-phase progression in cells after HU removal, due to efficient late-firing origins. Additionally, checkpoint kinase Cds1p is required for maintaining the efficient replication/firing late in S-phase. We further propose that efficient late-firing origins are essential for ensuring completion of DNA duplication by the end of S-phase.
Highlights
DNA replication is a key event in the cell cycle, occurring within a confined period termed S-phase
Microarray analysis of enriched heavy:light nascent DNA in Saccharomyces cerevisiae has revealed a genomic view of DNA replication timing profiles: some regions of the genome are always replicated early in S phase, some in the middle, and others at the end, due to a strict timing of firing at origins [4]
We show that the rate of DNA copy number-increase in cells released after HU-arrest, is generally slow in early S-phase due to the inefficient early-firing at origins
Summary
DNA replication is a key event in the cell cycle, occurring within a confined period termed S-phase. Studies in human HeLa cells have shown that more than 60% of the genome is slowly replicated throughout the S-phase, owing to a flexible timing of firing or inefficient firing at origins [7]. It has been shown that DNA replication/firing in S. pombe is less efficient than that in S. cerevisiae [8,9]. During S. pombe S-phase, initiation of DNA replication occurs at multiple sites (origins) that are enriched with AT-rich sequences, at various times. Using the genome-wide ORF-specific DNA microarray analysis, we show that in S. pombe, individual origins fire with varying efficiencies and at different times during S-phase. Our results indicate that efficient DNA replication/firing occurs late in S-phase progression in cells after HU removal, due to efficient late-firing origins.
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