Abstract
Chromosome replication in Saccharomyces cerevisiae is initiated from ~300 origins that are regulated by DNA sequence and by the limited abundance of six trans-acting initiation proteins (Sld2, Sld3, Dpb11, Dbf4, Sld7 and Cdc45). We set out to determine how the levels of individual factors contribute to time of origin activation and/or origin efficiency using induced depletion of single factors and overexpression of sets of multiple factors. Depletion of Sld2 or Sld3 slows growth and S phase progression, decreases origin efficiency across the genome and impairs viability as a result of incomplete replication of the rDNA. We find that the most efficient early origins are relatively unaffected by depletion of either Sld2 or Sld3. However, Sld3 levels, and to a lesser extent Sld2 levels, are critical for firing of the less efficient early origins. Overexpression of Sld3 simultaneously with Sld2, Dpb11 and Dbf4 preserves the relative efficiency of origins. Only when Cdc45 and Sld7 are also overexpressed is origin efficiency equalized between early- and late-firing origins. Our data support a model in which Sld3 together with Cdc45 (and/or Sld7) is responsible for the differential efficiencies of origins across the yeast genome.
Highlights
One of the enduring mysteries in the field of eukaryotic DNA replication is the variability in initiation of DNA replication across a genome
A conserved property of eukaryotic origins is that they vary in efficiency—the proportion of cells in a population in which they “fire”—and in the average time of activation within S phase, but the molecular details underlying this variation are not well understood
Previous work has shown that limiting concentrations of a set of conserved replication initiation proteins referred to as “SSDDCS” (Sld2, Sld3, Dbf4, Dpb11, Cdc45, and Sld7) are rate limiting for origin activation in budding yeast and other eukaryotes; combined overexpression of these proteins increases and/or advances origin firing
Summary
One of the enduring mysteries in the field of eukaryotic DNA replication is the variability in initiation of DNA replication across a genome. Studies of DNA replication revealed that certain parts of the eukaryotic genome are replicated earlier in S phase than others, and that the large-scale organization of chromosomes, their underlying sequence and chromatin structure play a regulatory role in this variability [3, 4] These differences in replication time arise from staggered origin firing during S phase with initiation occurring in a continuum from early to later in S phase [5, 6]. Because efficiency is the outcome of the interplay between origin competence and extrinsic variables such as location of nearby origins and their properties, measuring the competence of individual origins has been difficult In broad terms, these two modes of variability in replication initiation, origin efficiency and origin firing time, are conserved from Saccharomyces cerevisiae to humans [6, 11]. The conservation of these features implies that plasticity in origin activation is biologically relevant, the nature of its importance for genome function is not entirely clear
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