Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide EMSA (gEMSA) of ORC and yeast genomic DNA

ABSTRACT: Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal DNA, triggering a series of molecular events that culminate in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome requires multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern with relevance to differentiation, genome stability and evolution. It is unclear whether ORC-origin interactions are relevant to the regulation of origin activation time. Here we applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as ‘chromatin-dependent’ when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. The two classes of origins were distinct in terms of local nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at ‘chromatin-dependent’ origins. Importantly, origins that fired in late S-phase, but not those that fired in early S-phase, showed a significant dependence on the ORC-origin DNA interaction for ORC binding in vivo. Therefore we demonstrate for the first time a connection between ORC-origin binding mechanisms and the regulation of origin activation time at a genome-wide level. Analysis of ORC genomic DNA binding across three ORC concentrations.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Catherine Fox

PROVIDER: E-GEOD-48440 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Eukaryotic DNA replication initiates from multiple sites on each chromosome called replication origins. In the budding yeast Saccharomyces cerevisiae, origins are defined at discrete sites. Regular spacing and diverse firing characteristics of origins are thought to be required for efficient completion of replication, especially in the presence of replication stress. However, a S. cerevisiae chromosome III harboring multiple origin deletions has been reported to replicate relatively normally, and yet how an origin-deficient chromosome could accomplish successful replication remains unkown. To address this issue, we deleted seven well-characterized origins from chromosome VI, and found that thsese deletions do not cause gross growth defects even in the presence of replication inhibitors. We demonstrated that the origin deletions do cause a strong decrease in the binding of the origin recognition complex. Unexpectedly, replication profiling of this chromosome showed that DNA replication initiates from non-canonical loci around deleted origins in yeast. These results suggest that replication initiation can be unexpectedly flexible in this organism. In this study, we aimed to establish an independent system to investigate how an origin-deficient chromosome is replicated. To this end, we systematically deleted seven well-characterized origins on the left arm of S. cerevisiae chromosome VI and analyzed (1) Orc2 localization during G2/M arrest and (2) BrdU incorporation during synchronous release from G1 arrest into S-phase, and compared the results to wild-type cell signals. For Orc2 ChIP-Chip experiments, Orc-bound DNA was isolated from Orc2-2Xlinker-3XFlag epitope-tagged cells arrested in G2/M using antibodies against Flag. For BrdU ChIP-Chip experiments, actively replicating DNA was isolated from cells harboring a single integrated BrdU incorporation vector released synchronously into 200mM HU using antibodies against BrdU. Immunoprecipitated and input (Orc2) or G1 (BrdU) DNA was then amplified and competitively hybridized to high-resolution strand-specific microarrays covering chromosomes III, VI, and XII.

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Genome-wide EMSA (gEMSA) of ORC and yeast genomic DNA

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