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, 9 (1), 9946

Telomere DNA Length-Dependent Regulation of DNA Replication Timing at Internal Late Replication Origins

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Telomere DNA Length-Dependent Regulation of DNA Replication Timing at Internal Late Replication Origins

Yudai Hasegawa et al. Sci Rep.

Abstract

DNA replication is initiated at replication origins on chromosomes at their scheduled time during S phase of the cell cycle. Replication timing control is highly conserved among eukaryotes but the underlying mechanisms are not fully understood. Recent studies have revealed that some telomere-binding proteins regulate replication timing at late-replicating origins throughout the genome. To investigate the molecular basis of this process, we analyzed the effects of excessive elongation of telomere DNA on replication timing by deleting telomere-associated shelterin proteins in Schizosaccharomyces pombe. We found that rap1∆ and poz1∆ cells showed abnormally accelerated replication at internal late origins but not at subtelomere regions. These defects were suppressed by removal of telomere DNA and by deletion of the telomere-binding protein Taz1. Furthermore, Sds21-a counter protein phosphatase against Dbf4-dependent kinase (DDK)-accumulated at elongated telomeres in a Taz1-dependent manner but was depleted at internal late origins, indicating that highly elongated telomeres sequester Sds21 at telomeres and perturb replication timing at internal regions. These results demonstrate that telomere DNA length is an important determinant of replication timing at internal regions of chromosomes in eukaryotes.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Rap1 deletion accelerates DNA replication from internal late origins. (A) Schematic illustration of telomere-binding proteins in S. pombe. (B) Replication timing of late origins is affected by Rap1 deletion. The locations of replication origins on chromosome 2 are schematically illustrated at the top. AT2024 and ars2004 are early origins (red squares), and AT2035, AT2080, AT2088, ars727, tel-60.0, and tel-0.2 are late origins (blue squares). The subtelomeric late origins tel-60.0 and tel-0.2 are located at 60.0 and 0.2 kb, respectively, from the telomere of the right arm of chromosome 2. The non-origin region (non-ori) is located at 30 kb from ars2004. Wild type, taz1∆, rap1∆, and rif1∆ cells were grown in EMM medium and released from G2/M arrest, and their DNA was labeled with BrdU for 90 min in the presence of 10 mM HU, purified by immunoprecipitation, and analyzed by qPCR. Each value was normalized to that of ars2004. Error bars indicate the s.d. (n = 3). Asterisks indicate a significant change as compared with the wild-type strain (p < 0.05, Student’s t-test).
Figure 2
Figure 2
The rap1∆ strain shows replication timing defects in a telomere DNA-dependent manner. The rap1trt1∆ strain showed replication timing similar to that observed in the trt1∆ strain at internal late origins in the absence of telomere DNA, whereas the rif1trt1∆ strain showed accelerated replication at late origins. Cells with circular chromosomes were grown in YES medium, and the BrdU incorporation assays were performed as in Fig. 1B. Each value was normalized to that of ars2004 and then to the trt1∆ value.
Figure 3
Figure 3
Replication timing defects in rap1∆ and poz1∆ are Taz1-dependent. The poz1∆ strain exhibited replication timing defects similar to those observed in the rap1∆ strain. These defects were suppressed by Taz1 deletion. The BrdU incorporation assays were performed as in Fig. 1B. Asterisks indicate a significant change as compared with the wild-type strain (p < 0.05, Student’s t-test).
Figure 4
Figure 4
Sds21 is sequestered to excessively elongated telomeres in a Taz1-dependent manner. (A) Localization of Rif1 at ars727 was unaffected by Rap1 or Poz1 deletion. ChIP analyses of Rif1-12Myc localization were performed with cells at 60 min after released from G2/M arrest. Co-purified genomic DNA was analyzed by qPCR. Each value was normalized to that of ars2004. Error bars indicate the s.d. (n = 3). Asterisks indicate a significant change as compared with the wild-type strain (p < 0.05, Student’s t-test). (B) Sds21 localization at ars727 was decreased by Rap1 deletion. ChIP analyses of Sds21-3Flag were performed with cells at 60 min after released from G2/M arrest. Each value was normalized to that of ars2004. Error bars indicate the s.d. (n = 3). Asterisks indicate a significant change as compared with the wild-type strain (p < 0.05, Student’s t-test). (C) Sds21 accumulates at telomeres in rap1∆ cells. DNA co-precipitated with Sds21-3Flag was analyzed by Southern blotting using telomere and rDNA (control) probes. Left panel, representative images cropped from the same Southern blots (the original blots are displayed in Supplemental Fig. S3). Each signal compared with its background was analyzed by ImageJ software (NIH, Bethesda, MD, USA) and was normalized to that of rDNA and then to the wild-type value (right). Error bars indicate the s.d. (n = 3). Asterisks indicate a significant change as compared with the wild-type strain (p < 0.05, Student’s t-test). (D) Localization of Sds21 at ars727 in rap1∆ cells was restored by Taz1 deletion. ChIP was performed as in (B). Asterisks indicate a significant change between the indicated strains (p < 0.05, Student’s t-test).
Figure 5
Figure 5
Overexpression of Sds21 causes severe defects in cell cycle progression. (A) Expression level of Sds21-3Flag protein in each strain. Wild type, Sds21-overexpressing wild type (wild type-Sds21OE), rap1∆, and Sds21-overexpressing rap1∆ (rap1∆-Sds21OE) cells were grown in EMM. The whole-cell extracts were analyzed by immunoblotting using anti-Flag (M2 F3165; Sigma-Aldrich) for Sds21-3Flag and anti-PSTAIRE (P7962; Sigma-Aldrich) for Cdc2 (loading control). (B) Representative septation index after release from G2/M block. (C) Wild type, wild type-Sds21OE, rap1∆, and rap1∆-Sds21OE cells were grown in EMM medium, and ChIP analyses of Sds21-3Flag localization were performed with cells in early S phase, i.e., at 60 min (for wild type and rap1∆), 130 min (for wild type-Sds21OE), and 80 min (for rap1∆-Sds21OE) after released from G2/M arrest. Co-purified genomic DNA was analyzed by qPCR. Each value was normalized to that of ars2004. Error bars indicate the s.d. (n = 3).
Figure 6
Figure 6
Model of replication timing control based on telomere DNA length. (A) In wild-type cells, the PP1 Sds21 is recruited to telomeres and late origins such as ars727, a Taz1-independent origin, through association with Rif1. Localization of Sds21 at ars727 maintains replication timing at this site in late S phase. Rif1 associates with telomeres via Taz1. (B) In rap1∆ cells, telomere DNA is excessively elongated, resulting in the accumulation of Sds21 via Taz1–Rif1 complexes. Sequestration of Sds21 at telomeres reduces Sds21 localization at ars727 and accelerates replication timing in early S phase. (C) In taz1rap1∆ cells, Sds21 is released from elongated telomeres, which restores Sds21 localization at ars727 and normal replication timing in late S phase. In contrast, the absence of Sds21 at telomeres accelerates replication timing at tel-0.2.

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