Linear Derivatives of Saccharomyces cerevisiae Chromosome III Can Be Maintained in the Absence of Autonomously Replicating Sequence Elements

Abstract

Replication origins in Saccharomyces cerevisiae are spaced at intervals of approximately 40 kb. However, both measurements of replication fork rate and studies of hypomorphic alleles of genes encoding replication initiation proteins suggest the question of whether replication origins are more closely spaced than should be required. We approached this question by systematically deleting replicators from chromosome III. The first significant increase in loss rate detected for the 315-kb full-length chromosome occurred only after all five efficient chromosomal replicators in the left two-thirds of the chromosome (ARS305, ARS306, ARS307, ARS309, and ARS310) had been deleted. The removal of the inefficient replicator ARS308 from this originless region caused little or no additional increase in loss rate. Chromosome fragmentations that removed the normally inactive replicators on the left end of the chromosome or the replicators distal to ARS310 on the right arm showed that both groups of replicators contribute significantly to the maintenance of the originless chromosome. Surprisingly, a 142-kb derivative of chromosome III, lacking all sequences that function as autonomously replicating sequence elements in plasmids, replicated and segregated properly 97% of the time. Both the replication initiation protein ORC and telomeres or a linear topology were required for the maintenance of chromosome fragments lacking replicators.In eukaryotes, DNA replication initiates at specific sites called replication origins. cis-acting sequences called replicators define the positions and regulate the activity of replication origins by promoting the assembly of prereplicative complexes (pre-RCs) during the G 1 phase of the cell cycle. Eukaryotic replicators were first identified in the budding yeast Saccharomyces cerevisiae on the basis of their ability to promote the extrachromosomal maintenance of plasmids (22, 50). The dissection of these autonomously replicating sequence (ARS) elements revealed an essential 11-bp sequence, called the ARS consensus sequence (ACS), that is required for both plasmid and chromosomal replicator activity (reviewed in reference 34). The ACS is the core of the binding site for the highly conserved six-subunit origin recognition complex (ORC), which recruits and assembles the other components of the pre-RC (reviewed in reference 1). Like the ORC, the other components of the pre-RC are highly conserved throughout the eukaryotic kingdom.Upon entry into S phase, pre-RCs are activated to initiate replication according to a temporal program whose determinants are poorly understood. The activation of replication origins requires the activity of two kinases, a cyclin-dependent kinase composed of the catalytic subunit encoded by CDC28 and regulatory subunits encoded by CLB5 and CLB6, and the Dbf4-dependent kinase, composed of a catalytic subunit encoded by CDC7 and a regulatory subunit encoded by DBF4. During replication initiation, DNA is unwound at origins and replication fork proteins are assembled to form repl...

Maintaining replication origins in the face of genomic change

“…Combined with knowledge that, when origins are lost, cryptic, ''backup'' origins may be able to fire (Dershowitz et al 2007;Blow et al 2011), our work suggests that the genome is littered with weak, potential origin ''seeds.'' In support of this idea, both the S. cerevisiae and L. waltii genomes have over 10,000 matches to the ACS sequence.…”

“…Unlike genes, any individual origin is believed to be redundant with neighboring origins and therefore nonessential (Dershowitz et al 2007). For these reasons, it is unclear what the selective forces might be that act to preserve any single origin over evolutionary time.…”

“…The essentiality of DNA replication is reflected in the extraordinary conservation of the replication machinery across eukaryotes (Sclafani and Holzen 2007), the multilayered regulation of DNA replication ensuring that the entire genome is replicated only once during S phase (Blow and Dutta 2005;Truong and Wu 2011), and the numerous DNA repair and checkpoint pathways that engage at any flag of error (Sclafani and Holzen 2007). However, origins of replication-the sites where replication is initiated-vary across eukaryotes (Sclafani and Holzen 2007;Masai et al 2010), are not all used in every cell cycle, and can be removed individually, as well as in large groups, without loss of cell viability (Dershowitz et al 2007). This contrasting duality-collectively being essential for life, while individually free to diverge-raises the question of how, or if, origins of replication are conserved over evolutionary time.…”

DNA Replication Origin Function Is Promoted by H3K4 Di-methylation inSaccharomyces cerevisiae

“…Also analogous to transcriptional control at promoters, we propose that histone H3K4 di-methylation is an important element of origin chromatin structure, but its absence alone does not severely inhibit origin function. In fact, origin activity is robust enough that even substantial reductions in expression of MCM proteins or deletion of many origins from a yeast chromosome causes no growth defects (Dershowitz et al 2007;Ge et al 2007;Blow et al 2011). Nevertheless, the effect of H3K4 dimethylation loss over several cell cycles or in combination with other replication perturbations causes a significant loss of replication fitness.…”

High-resolution mapping, characterization, and optimization of autonomously replicating sequences in yeast

“…While these studies are useful in detailing the temporal order of events during genome replication, they fall short of generating a complete map of potential origin sites, due to low resolution and variability in origin usage and efficiency in different cells within a population. Deletion of all known active origin sites on a chromosome does not completely abrogate replication (Dershowitz et al 2007), suggesting the presence of cryptic origins whose chromosomal replication initiation signal is too weak to be detected in population-based assays. Thus, ARS mapping and functional dissection remain the most precise tools for understanding the molecular determinants of yeast origin function.…”