The spatial arrangement of ORC binding modules determines the functionality of replication origins in budding yeast

Department of Biochemistry, Molecular Biology and Biophysics, 6-155 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA.
Nucleic Acids Research (Impact Factor: 9.11). 02/2006; 34(18):5069-80. DOI: 10.1093/nar/gkl661
Source: PubMed


In the quest to define autonomously replicating sequences (ARSs) in eukaryotic cells, an ARS consensus sequence (ACS) has
emerged for budding yeast. This ACS is recognized by the replication initiator, the origin recognition complex (ORC). However,
not every match to the ACS constitutes a replication origin. Here, we investigated the requirements for ORC binding to origins
that carry multiple, redundant ACSs, such as ARS603. Previous studies raised the possibility that these ACSs function as individual ORC binding sites. Detailed mutational analysis
of the two ACSs in ARS603 revealed that they function in concert and give rise to an initiation pattern compatible with a single bipartite ORC binding
site. Consistent with this notion, deletion of one base pair between the ACS matches abolished ORC binding at ARS603. Importantly, loss of ORC binding in vitro correlated with the loss of ARS activity in vivo. Our results argue that replication origins in yeast are in general comprised of bipartite ORC binding sites that cannot
function in random alignment but must conform to a configuration that permits ORC binding. These requirements help to explain
why only a limited number of ACS matches in the yeast genome qualify as ORC binding sites.

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Available from: Anja Katrin Bielinsky, Mar 20, 2014
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    • "Our results show that mutating the original ACS abolishes the ARS function of ARS1, but does not abolish the ARS function of ARS1max. This finding suggests that ARS1max may be a dimeric ARS (Bolon and Bielinsky 2006). "
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    ABSTRACT: DNA replication origins are necessary for the duplication of genomes. In addition, plasmid-based expression systems require DNA replication origins to efficiently maintain plasmids. The yeast Autonomously Replicating Sequence (ARS) assay has been a valuable tool in dissecting replication origin structure and function. However, the dearth of information on origins in diverse yeasts limits the availability of efficient replication origin modules to only a handful of species and restricts our understanding of origin function and evolution. To enable rapid study of origins we have developed a sequencing-based suite of methods for comprehensively mapping and characterizing ARSs within a yeast genome. Our approach finely maps genomic inserts capable of supporting plasmid replication and uses massively parallel deep mutational scanning to define molecular determinants of ARS function with single nucleotide resolution. In addition to providing unprecedented detail into origin structure, our data have allowed us to design short, synthetic DNA sequences that retain maximal ARS function. These methods can be readily applied to understand and modulate ARS function in diverse systems.
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    • "At present we do not know whether this extended ORC domain represents binding of ORC to different sites on different DNA strands or binding of multiple ORCs on the same DNA strand. The possibility that multiple ORCs bind a single DNA strand is consistent with previous evidence for increased ORC occupancy on longer origin fragments in vitro and the binding of multiple ORCs at some origins in yeast and metazoa in vivo (Bielinsky et al., 2001; Takahashi et al., 2003; Bolon and Bielinsky, 2006; Aladjem, 2007). However, the functional significance of multiple ORCs for origin activity or initiation site selection remains unclear. "
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    ABSTRACT: Epigenetic regulation exerts a major influence on origins of DNA replication during development. The mechanisms for this regulation, however, are poorly defined. We showed previously that acetylation of nucleosomes regulates the origins that mediate developmental gene amplification during Drosophila oogenesis. Here we show that developmental activation of these origins is associated with acetylation of multiple histone lysines. Although these modifications are not unique to origin loci, we find that the level of acetylation is higher at the active origins and quantitatively correlated with the number of times these origins initiate replication. All of these acetylation marks were developmentally dynamic, rapidly increasing with origin activation and rapidly declining when the origins shut off and neighboring promoters turn on. Fine-scale analysis of the origins revealed that both hyperacetylation of nucleosomes and binding of the origin recognition complex (ORC) occur in a broad domain and that acetylation is highest on nucleosomes adjacent to one side of the major site of replication initiation. It was surprising to find that acetylation of some lysines depends on binding of ORC to the origin, suggesting that multiple histone acetyltransferases may be recruited during origin licensing. Our results reveal new insights into the origin epigenetic landscape and lead us to propose a chromatin switch model to explain the coordination of origin and promoter activity during development.
    Preview · Article · Nov 2011 · Molecular biology of the cell
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    • "ORC was purified from baculovirus-infected Sf9 cells as described (26). The ORC-ARS EMSA were performed as described (27) with some modifications. Biotin-labeled specific DNA probes (102 bp) of ARS1, ARS606 and ARS607 were generated by PCR using the primers 5′-biotin- AAAGCCAAATGATTTAGCAT-3′/5′-GTGCACTTGCCTGCAGGCCT-3′ for ARS1, 5′-biotin-CATCCTCAATCATCAATTAA-3′/5′-GGGCGCTTTTTTTGTGACAT-3′ for ARS606, 5′-biotin- ACACTACATTCGCTAAATTAC-3′/5′-TGGTCACTCCAGATCTAGTTT-3′ for ARS607 (IDT). "
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    ABSTRACT: In budding yeast, the eukaryotic initiator protein ORC (origin recognition complex) binds to a bipartite sequence consisting of an 11 bp ACS element and an adjacent B1 element. However, the genome contains many more matches to this consensus than actually bind ORC or function as origins in vivo. Although ORC-dependent loading of the replicative MCM helicase at origins is enhanced by a distal B2 element, less is known about this element. Here, we analyzed four highly active origins (ARS309, ARS319, ARS606 and ARS607) by linker scanning mutagenesis and found that sequences adjacent to the ACS contributed substantially to origin activity and ORC binding. Using the sequences of four additional B2 elements we generated a B2 multiple sequence alignment and identified a shared, degenerate 8 bp sequence that was enriched within 228 known origins. In addition, our high-resolution analysis revealed that not all origins exist within nucleosome free regions: a class of Sir2-regulated origins has a stably positioned nucleosome overlapping or near B2. This study illustrates the conserved yet flexible nature of yeast origin architecture to promote ORC binding and origin activity, and helps explain why a strong match to the ORC binding site is insufficient to identify origins within the genome.
    Full-text · Article · May 2011 · Nucleic Acids Research
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