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. 2010 Apr 15;24(8):748-53.
doi: 10.1101/gad.1913210. Epub 2010 Mar 29.

Conserved Nucleosome Positioning Defines Replication Origins

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Free PMC article

Conserved Nucleosome Positioning Defines Replication Origins

Matthew L Eaton et al. Genes Dev. .
Free PMC article

Abstract

The origin recognition complex (ORC) specifies replication origin location. The Saccharomyces cerevisiae ORC recognizes the ARS (autonomously replicating sequence) consensus sequence (ACS), but only a subset of potential genomic sites are bound, suggesting other chromosomal features influence ORC binding. Using high-throughput sequencing to map ORC binding and nucleosome positioning, we show that yeast origins are characterized by an asymmetric pattern of positioned nucleosomes flanking the ACS. The origin sequences are sufficient to maintain a nucleosome-free origin; however, ORC is required for the precise positioning of nucleosomes flanking the origin. These findings identify local nucleosomes as an important determinant for origin selection and function.

Figures

Figure 1.
Figure 1.
Precise localization of yeast origins. (A) Read depth of ORC-enriched sequence tags from chromosome XIV from G2-arrested cells. ORC-binding sites identified in this study (blue) and a prior study (green) (Xu et al. 2006) are indicated by triangles. (B) Venn diagram of the overlap between the 267 ORC peaks identified in this study (blue) and the 396 areas of ORC enrichment defined in Xu et al. (2006) (green) (see the Supplemental Material). Of the ORC peaks identified in this study, 258 overlapped 241 peaks from the previous study. (C) The position weight matrix of the ORC binding consensus built from the ORC–ACS set. Each nucleotide is represented by a discrete color (red [T], orange [G], blue [C], and green [A]).
Figure 2.
Figure 2.
Replication origins are associated with an asymmetric NFR flanked by well-positioned nucleosomes. (A) Heat map of nucleosome occupancy and average nucleosome signal from asynchronous cells for 219 ORC–ACS sites. Interpreted nucleosome positions are represented as ovals. (B) Nucleosome occupancy at 238 nr-ACS sites. Vertical dashed lines represent the center of the 33-bp ACS motif match. All ACS matches are oriented relative to the T-rich strand and are aligned at position 0.
Figure 3.
Figure 3.
Sequence polarity at replication origins. (A) Schematic of nucleosome positioning at the prototypical ARS1 replication origin. The positions of the ACS and B elements relative to the adjacent nucleosomes are indicated. (B) Heat map of nucleosome occupancy from asynchronous cells at the ORC–ACS sites. Red and green dots represent the bias in nucleotide composition (T-rich or A-rich, respectively) at each sequence (see the Materials and Methods). Histograms above the heat map show the cumulative incidence of T-rich or A-rich windows (red and green, respectively) over the region in 20-bp windows. (C) Nucleosome occupancy and sequence bias for the nr-ACS set.
Figure 4.
Figure 4.
ORC is necessary and sufficient for precise nucleosome positioning. (A) Heat map of nucleosome occupancy around ORC–ACS sites in wild-type cells arrested in G2 at 37°C. (B) Heat map of nucleosome occupancy around ORC–ACS sites in orc1-161 cells arrested in G2 at 37°C. (C) Density of wild-type (black) and orc1-161 (red) nucleosome dyads surrounding ORC–ACS sites arrested in G2 at 37°C. (D) The density of nucleosome dyads assembled in vitro at ARS1 in the presence or absence of ORC.

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