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Chromatin-dependent and -independent regulation of DNA replication origin activation in budding yeast - PubMed

Chromatin-dependent and -independent regulation of DNA replication origin activation in budding yeast

Marko Lõoke et al. EMBO Rep. 2013 Feb.

Abstract

To elucidate the role of the chromatin environment in the regulation of replication origin activation, autonomously replicating sequences were inserted into identical locations in the budding yeast genome and their activation times in S phase determined. Chromatin-dependent origins adopt to the firing time of the surrounding locus. In contrast, the origins containing two binding sites for Forkhead transcription factors are activated early in the S phase regardless of their location in the genome. Our results also show that genuinely late-replicating parts of the genome can be converted into early-replicating loci by insertion of a chromatin-independent early replication origin, ARS607, whereas insertion of two Forkhead-binding sites is not sufficient for conversion.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1

Recruitment of Cdc45 and Pol2 to replication origins. (A) DNA content analysis of AKY543 strain at indicated time-points after release from G1 arrest. (B–H) Recruitment of Cdc45 and Pol2 proteins to various replication origins throughout the S phase. The relative binding of the factors to the origins at indicated time-points are shown, 1 was defined as the average value of unbound samples. Error bars indicate s.d. of three experiments. ARS, autonomously replicating sequence.

Figure 2
Figure 2

Synchronization of chromatin-dependent origins in the VPS13 locus. Relative binding of Cdc45 to origins is shown. (A) VPS13-ARS409 and ARS409; (B) VPS13-ARS501 and ARS501; (C) VPS13-ARS605 and ARS605; (D) VPS13-ARS609 and ARS609. The binding of Cdc45 to the origin-free VPS13 locus is shown for reference in all panels. Error bars indicate s.d. of three experiments. ARS, autonomously replicating sequence.

Figure 3
Figure 3

Location-independent activation of early origins in the VPS13 locus. Relative binding of Cdc45 to origins is shown. (A) VPS13-ARS305 and ARS305; (B) VPS13-ARS607 and ARS607; (C) VPS13-ARS737 and ARS737. The binding of Cdc45 to the origin-free VPS13 locus is shown for reference in all panels. Error bars indicate s.d. of three experiments. ARS, autonomously replicating sequence.

Figure 4
Figure 4

Activation of ARS607 in ectopic loci. (A) Schematic representation of ARS305, ARS737, ARS607 and their mutants. Deletions of ARS607 and the sites of point mutations of all origins are shown. Sequences corresponding to the Fkh1/2 consensus sequence RTAAAYA [33] are indicated as grey boxes, ACS is shown in bold, B3 and B4 boxes in ARS607 [7] and ARS305 [3] are underlined. (B–I) Relative binding of Cdc45 to indicated wild-type and mutated ARS sequences in the VPS13 locus. The binding of Cdc45 to the origin-free VPS13 is shown for reference in all panels. (J) Activation of ARS607 in different genomic loci. Relative binding of the Cdc45 protein to the ARS607 sequence inserted into VPS13, DPB11, HXK1 or CLD1 loci was determined. The binding of Cdc45 to genuine ARS607 and origin-free VPS13, HXK1 and CLD1 loci is shown for reference. Error bars indicate s.d. of three experiments. ARS, autonomously replicating sequence; mut, mutation.

Figure 5
Figure 5

Binding of Fkh1 to ARS loci. (A) Relative binding of Fkh1 protein to different native replication origins and mutants of ARS607, ARS305 and ARS737 in VPS13 locus was determined by ChIP assay in G1-arrested cells. ChIP signal from the origin-free VPS13 locus was set to 1. Error bars indicate s.d. of three experiments. (B) Box plots of replication indexes of budding yeast ARS sequences containing 0–2 binding sites for Forkhead factors. Origins containing two Fkh1/2 sites at various distances from each other were analysed. T-test P-values for the difference in the means between all origins and each selected group are shown. ARS, autonomously replicating sequence.

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References

    1. Marahrens Y, Stillman B (1992) A yeast chromosomal origin of DNA replication defined by multiple functional elements. Science 255: 817–823 - PubMed
    1. Rao H, Stillman B (1995) The origin recognition complex interacts with a bipartite DNA binding site within yeast replicators. Proc Natl Acad Sci USA 92: 2224–2228 - PMC - PubMed
    1. Huang RY, Kowalski D (1996) Multiple DNA elements in ARS305 determine replication origin activity in a yeast chromosome. Nucl Acids Res 24: 816–823 - PMC - PubMed
    1. Lin S, Kowalski D (1997) Functional equivalency and diversity of cis-acting elements among yeast replication origins. Mol Cell Biol 17: 5473–5484 - PMC - PubMed
    1. Speck C, Stillman B (2007) Cdc6 ATPase activity regulates ORC x Cdc6 stability and the selection of specific DNA sequences as origins of DNA replication. J Biol Chem 282: 11705–11714 - PMC - PubMed

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