Origin firing at repetitive sequences and genome replication

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PROJECT SUMMARY/ABSTRACT Over half of the human genome is comprised of repetitive DNA sequences organized as gene-poor, late-replicating, transcriptionally silent heterochromatin. Recent studies have discerned widespread transcriptional de-repression at repetitive regions during carcinogenesis and aging. This de-repression accelerates replication of these regions, which normally replicate late, thereby depleting limiting pools of replication resources and compromising replication in gene-rich transcriptionally active chromatin. Despite the importance and prevalence of the association between low levels of transcription and late replication at repetitive sequences, the mechanistic basis for this link remains unclear. The ribosomal DNA (rDNA) and the copper-inducible CUP1 arrays in budding yeast provide powerful experimental systems in which to elucidate these mechanisms: First, at each locus, a single manipulation, Sir2 depletion at the rDNA, and copper administration at CUP1, activates both transcription and replication, providing a simple tool to manipulate both processes. Second, each rDNA and CUP1 repeat contains a single, sequence-defined origin of replication; this creates uniform and predictable positioning of pre-replicative complexes (pre-RC), which are required for initiation of DNA replication, and nucleosomes, which define the chromatin context in which these pre-RCs must act. We have developed sequencing-based methods that reveal the precise locations of pre-RCs and nucleosomes, both at these repetitive arrays and at unique origins across the genome. Using these methods, we have discovered a feature of the chromatin at both the rDNA and CUP1 origins that may mechanistically link the processes of transcription and replication: In the absence of transcription, the pre-RCs at both origins are closely flanked by precisely-positioned nucleosomes, while transcription decreases nucleosome occupancy at these sites and activates replication. Using this experimental setup and the tools for chromatin profiling we have developed, we will determine (1) whether high nucleosome occupancy adjacent to pre-RC inhibits replication initiation and (2) how nucleosome remodeling enzymes, which we have shown to be required for transcription-induced replication at the rDNA array, relieve this nucleosome-imposed constraint and activate replication origins.