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Centromere identity in Drosophila is not determined in vivo by replication timing.

Sullivan B, Karpen G - J. Cell Biol. (2001)

Bottom Line: Minichromosomes with structurally intact centromeres were replicated in late S phase, and those in which centric and surrounding heterochromatin were partially or fully deleted were replicated earlier in mid S phase.We provide the first in vivo evidence that centromeric chromatin is replicated at different times in S phase.These studies indicate that incorporation of CID/CENP-A into newly duplicated centromeres is independent of replication timing and argue against determination of centromere identity by temporal sequestration of centromeric chromatin replication relative to bulk genomic chromatin.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.

ABSTRACT
Centromeric chromatin is uniquely marked by the centromere-specific histone CENP-A. For assembly of CENP-A into nucleosomes to occur without competition from H3 deposition, it was proposed that centromeres are among the first or last sequences to be replicated. In this study, centromere replication in Drosophila was studied in cell lines and in larval tissues that contain minichromosomes that have structurally defined centromeres. Two different nucleotide incorporation methods were used to evaluate replication timing of chromatin containing CID, a Drosophila homologue of CENP-A. Centromeres in Drosophila cell lines were replicated throughout S phase but primarily in mid S phase. However, endogenous centromeres and X-derived minichromosome centromeres in vivo were replicated asynchronously in mid to late S phase. Minichromosomes with structurally intact centromeres were replicated in late S phase, and those in which centric and surrounding heterochromatin were partially or fully deleted were replicated earlier in mid S phase. We provide the first in vivo evidence that centromeric chromatin is replicated at different times in S phase. These studies indicate that incorporation of CID/CENP-A into newly duplicated centromeres is independent of replication timing and argue against determination of centromere identity by temporal sequestration of centromeric chromatin replication relative to bulk genomic chromatin.

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Molecularly and functionally defined centromeres are replicated in late S phase. Single labeling of larval neuroblasts containing Dp1187–derived minichromosomes (Fig. 2 B). Centromeres were identified using CID antibodies (red) and sites of replication by anti-BrdU antibodies (green). Gray-scale images (DAPI) identify the minichromosome. Line plots quantitated pixel intensities for each wavelength along the width of the centromere (line). (A) Dpγ238 (1.3 Mb) and endogenous X centromeres replicated within 2 h of mitosis when CID staining (arrow) colocalized with BrdU staining (graph). (B) Dp10B (720 kb) containing 420 kb of CEN DNA and no surrounding heterochromatin was replicated 1–2 h before mitosis. (C) DpJ21A (580 kb) containing only half of the CEN DNA replicated earlier (2–4 h before mitosis) than the larger heterochromatin-containing minichromosomes.
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fig3: Molecularly and functionally defined centromeres are replicated in late S phase. Single labeling of larval neuroblasts containing Dp1187–derived minichromosomes (Fig. 2 B). Centromeres were identified using CID antibodies (red) and sites of replication by anti-BrdU antibodies (green). Gray-scale images (DAPI) identify the minichromosome. Line plots quantitated pixel intensities for each wavelength along the width of the centromere (line). (A) Dpγ238 (1.3 Mb) and endogenous X centromeres replicated within 2 h of mitosis when CID staining (arrow) colocalized with BrdU staining (graph). (B) Dp10B (720 kb) containing 420 kb of CEN DNA and no surrounding heterochromatin was replicated 1–2 h before mitosis. (C) DpJ21A (580 kb) containing only half of the CEN DNA replicated earlier (2–4 h before mitosis) than the larger heterochromatin-containing minichromosomes.

Mentions: To test if heterochromatin restricts centromeric replication to late S phase, replication of five structurally distinct minichromosomes was also studied (Fig. 2 B). The centromere (CEN) of the parental minichromosome, Dp8-23, is surrounded by 400 kb of centric heterochromatin. Dpγ238 was generated by an inversion in Dp8-23 so that its CEN is oriented in the opposite direction and is flanked by euchromatin on one side and 600 kb of heterochromatin on the other (Murphy and Karpen, 1995b; Sun et al., 1997; Williams et al., 1998). Both Dp8-23 (unpublished data) and Dpγ238 (Fig. 3 A) showed complete BrdU incorporation at the centromere and over the entire chromosome late in S phase, 1–3 h before M. Dp1187 was derived from the endogenous X chromosome, and consistent with its origin, intact minichromosome centromeres replicated coincident with the endogenous X centromere (Fig. 3 A). Two deleted minichromosomes, Dp10B (Fig. 3B) and Dpγ1230, in which the only centric heterochromatin present corresponds to the functional centromere were completely labeled by BrdU in late S phase.


Centromere identity in Drosophila is not determined in vivo by replication timing.

Sullivan B, Karpen G - J. Cell Biol. (2001)

Molecularly and functionally defined centromeres are replicated in late S phase. Single labeling of larval neuroblasts containing Dp1187–derived minichromosomes (Fig. 2 B). Centromeres were identified using CID antibodies (red) and sites of replication by anti-BrdU antibodies (green). Gray-scale images (DAPI) identify the minichromosome. Line plots quantitated pixel intensities for each wavelength along the width of the centromere (line). (A) Dpγ238 (1.3 Mb) and endogenous X centromeres replicated within 2 h of mitosis when CID staining (arrow) colocalized with BrdU staining (graph). (B) Dp10B (720 kb) containing 420 kb of CEN DNA and no surrounding heterochromatin was replicated 1–2 h before mitosis. (C) DpJ21A (580 kb) containing only half of the CEN DNA replicated earlier (2–4 h before mitosis) than the larger heterochromatin-containing minichromosomes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196454&req=5

fig3: Molecularly and functionally defined centromeres are replicated in late S phase. Single labeling of larval neuroblasts containing Dp1187–derived minichromosomes (Fig. 2 B). Centromeres were identified using CID antibodies (red) and sites of replication by anti-BrdU antibodies (green). Gray-scale images (DAPI) identify the minichromosome. Line plots quantitated pixel intensities for each wavelength along the width of the centromere (line). (A) Dpγ238 (1.3 Mb) and endogenous X centromeres replicated within 2 h of mitosis when CID staining (arrow) colocalized with BrdU staining (graph). (B) Dp10B (720 kb) containing 420 kb of CEN DNA and no surrounding heterochromatin was replicated 1–2 h before mitosis. (C) DpJ21A (580 kb) containing only half of the CEN DNA replicated earlier (2–4 h before mitosis) than the larger heterochromatin-containing minichromosomes.
Mentions: To test if heterochromatin restricts centromeric replication to late S phase, replication of five structurally distinct minichromosomes was also studied (Fig. 2 B). The centromere (CEN) of the parental minichromosome, Dp8-23, is surrounded by 400 kb of centric heterochromatin. Dpγ238 was generated by an inversion in Dp8-23 so that its CEN is oriented in the opposite direction and is flanked by euchromatin on one side and 600 kb of heterochromatin on the other (Murphy and Karpen, 1995b; Sun et al., 1997; Williams et al., 1998). Both Dp8-23 (unpublished data) and Dpγ238 (Fig. 3 A) showed complete BrdU incorporation at the centromere and over the entire chromosome late in S phase, 1–3 h before M. Dp1187 was derived from the endogenous X chromosome, and consistent with its origin, intact minichromosome centromeres replicated coincident with the endogenous X centromere (Fig. 3 A). Two deleted minichromosomes, Dp10B (Fig. 3B) and Dpγ1230, in which the only centric heterochromatin present corresponds to the functional centromere were completely labeled by BrdU in late S phase.

Bottom Line: Minichromosomes with structurally intact centromeres were replicated in late S phase, and those in which centric and surrounding heterochromatin were partially or fully deleted were replicated earlier in mid S phase.We provide the first in vivo evidence that centromeric chromatin is replicated at different times in S phase.These studies indicate that incorporation of CID/CENP-A into newly duplicated centromeres is independent of replication timing and argue against determination of centromere identity by temporal sequestration of centromeric chromatin replication relative to bulk genomic chromatin.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.

ABSTRACT
Centromeric chromatin is uniquely marked by the centromere-specific histone CENP-A. For assembly of CENP-A into nucleosomes to occur without competition from H3 deposition, it was proposed that centromeres are among the first or last sequences to be replicated. In this study, centromere replication in Drosophila was studied in cell lines and in larval tissues that contain minichromosomes that have structurally defined centromeres. Two different nucleotide incorporation methods were used to evaluate replication timing of chromatin containing CID, a Drosophila homologue of CENP-A. Centromeres in Drosophila cell lines were replicated throughout S phase but primarily in mid S phase. However, endogenous centromeres and X-derived minichromosome centromeres in vivo were replicated asynchronously in mid to late S phase. Minichromosomes with structurally intact centromeres were replicated in late S phase, and those in which centric and surrounding heterochromatin were partially or fully deleted were replicated earlier in mid S phase. We provide the first in vivo evidence that centromeric chromatin is replicated at different times in S phase. These studies indicate that incorporation of CID/CENP-A into newly duplicated centromeres is independent of replication timing and argue against determination of centromere identity by temporal sequestration of centromeric chromatin replication relative to bulk genomic chromatin.

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