DNA Physical Parameters Modulate Nucleosome Positioning in the Saccharomyces cerevisiae Genome

The so-called 601 DNA sequence is often used to constrain the position of nucleosomes on a DNA molecule in vitro. Although the ability of the 147 base pair sequence to precisely position a nucleosome in vitro is well documented, in vivo application of this property has been explored only in a few studies and yielded contradictory conclusions. Our goal in the present study was to test the ability of the 601 sequence to dictate nucleosome positioning in Saccharomyces cerevisiae in the context of a long tandem repeat array inserted in a yeast chromosome. We engineered such arrays with three different repeat size, namely 167, 197 and 237 base pairs. Although our arrays are able to position nucleosomes in vitro as expected, analysis of nucleosome occupancy on these arrays in vivo revealed that nucleosomes are not preferentially positioned as expected on the 601-core sequence along the repeats and that the measured nucleosome repeat length does not correspond to the one expected by design. Altogether our results demonstrate that the rules defining nucleosome positions on this DNA sequence in vitro are not valid in vivo, at least in this chromosomal context, questioning the relevance of using the 601 sequence in vivo to achieve precise nucleosome positioning on designer synthetic DNA sequences.

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Purification and characterization of OBF1: a Saccharomyces cerevisiae protein that binds to autonomously replicating sequences

Molecular and Cellular Biology ◽

10.1128/mcb.9.7.2906-2913.1989 ◽

1989 ◽

Vol 9 (7) ◽

pp. 2906-2913

Author(s):

S C Francesconi ◽

S Eisenberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Binding ◽

Sodium Dodecyl ◽

Molecular Size ◽

Protein Band ◽

Binding Activity ◽

Physical Parameters ◽

Major Protein ◽

Dna Binding Activity ◽

Major Protein Band

We previously identified a protein activity from Saccharomyces cerevisiae, OBF1, that bound specifically to a DNA element present in autonomously replicating sequences ARS120 and ARS121 (S. Eisenberg C. Civalier, and B. K. Tye, Proc. Natl. Acad. Sci. USA 85:743-746, 1988). OBF1 has now been purified to near homogeneity by conventional protein and DNA affinity chromatography. Electrophoresis of the purified protein in sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two polypeptides. The major protein band had a relative molecular size of 123 kilodaltons, and the minor protein band, which constituted only a small fraction of total protein, had a molecular size of 127 kilodaltons. Both polypeptides cochromatographed with the specific ARS120 DNA-binding activity and formed a stable protein-DNA complex, isolatable by sedimentation through sucrose gradients. Using antibodies, we have shown that both polypeptides are associated with the isolated protein-DNA complexes. The ARS DNA-binding activity had a Stokes radius of 54 A (5.4 nm) and a sedimentation coefficient of 4.28S, as determined by gel filtration and sedimentation through glycerol gradients, respectively. These physical parameters, together with the denatured molecular size values, suggested that the proteins exist in solution as asymmetric monomers. Since both polypeptides recognized identical sequences and had similar physical properties, they are probably related. In addition to binding to ARS120, we found that purified OBF1 bounds with equal affinity to ARS121 and with 5- and 10-fold-lower affinity to ARS1 and HMRE, respectively. Furthermore, in the accompanying paper (S. S. Walker, S. C. Francesconi, B. K. Tye, and S. Eisenberg, Mol. Cell. Biol. 9:2914-2921, 1989), we demonstrate the existence of a high, direct correlation between the ability of purify OBF1 to bind to ARS121 and optimal in vivo ARS121 activity as an origin of replication. These findings, taken together, suggest a role for OBF1 in ARS function, presumably at the level of initiation of DNA replication at the ARS.

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Multiple nucleosome positioning with unique rotational setting for the Saccharomyces cerevisiae 5S rRNA gene in vitro and in vivo.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.90.20.9315 ◽

1993 ◽

Vol 90 (20) ◽

pp. 9315-9319 ◽

Cited By ~ 33

Author(s):

M. Buttinelli ◽

E. Di Mauro ◽

R. Negri

Keyword(s):

Saccharomyces Cerevisiae ◽

Nucleosome Positioning ◽

5S Rrna ◽

Rrna Gene ◽

5S Rrna Gene

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Nucleosome Positioning in Saccharomyces cerevisiae

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.00046-10 ◽

2011 ◽

Vol 75 (2) ◽

pp. 301-320 ◽

Cited By ~ 59

Author(s):

A. Jansen ◽

K. J. Verstrepen

Keyword(s):

Saccharomyces Cerevisiae ◽

Nucleosome Positioning

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Nucleosome positioning, nucleotide excision repair and photoreactivation in Saccharomyces cerevisiae

DNA Repair ◽

10.1016/j.dnarep.2015.09.012 ◽

2015 ◽

Vol 36 ◽

pp. 98-104 ◽

Cited By ~ 8

Author(s):

Laetitia Guintini ◽

Romain Charton ◽

François Peyresaubes ◽

Fritz Thoma ◽

Antonio Conconi

Keyword(s):

Saccharomyces Cerevisiae ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Nucleosome Positioning ◽

Nucleotide Excision

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Transcription, nucleosome positioning and protein binding modulate nucleotide excision repair of the Saccharomyces cerevisiae MET17 promoter

DNA Repair ◽

10.1016/s1568-7864(02)00239-2 ◽

2003 ◽

Vol 2 (4) ◽

pp. 375-386 ◽

Cited By ~ 15

Author(s):

N Powell

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Binding ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Nucleosome Positioning ◽

Nucleotide Excision

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Spt4 Facilitates the Movement of RNA Polymerase II through the +2 Nucleosomal Barrier

10.1101/2021.03.03.433772 ◽

2021 ◽

Author(s):

Ülkü Uzun ◽

Thomas Brown ◽

Harry Fischl ◽

Andrew Angel ◽

Jane Mellor

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Elongation Factor ◽

Nucleosome Positioning ◽

Gene Body ◽

Precise Position ◽

Transcription Elongation Factor

AbstractSpt4 is a transcription elongation factor, with homologues in organisms with nucleosomes. Structural and in vitro studies implicate Spt4 in transcription through nucleosomes, yet the in vivo function of Spt4 is unclear. Here we assessed the precise position of Spt4 during transcription and the consequences of loss of Spt4 on RNA polymerase II (RNAPII) dynamics and nucleosome positioning in Saccharomyces cerevisiae. In the absence of Spt4, the spacing between gene-body nucleosomes increases and RNAPII accumulates upstream of the nucleosomal dyad, most dramatically at nucleosome +2. Spt4 associates with elongating RNAPII early in transcription and its association dynamically changes depending on nucleosome positions. Together, our data show that Spt4 regulates early elongation dynamics, participates in co-transcriptional nucleosome positioning, and promotes RNAPII movement through the gene-body nucleosomes, especially the +2 nucleosome.

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Purification and characterization of OBF1: a Saccharomyces cerevisiae protein that binds to autonomously replicating sequences.

Molecular and Cellular Biology ◽

10.1128/mcb.9.7.2906 ◽

1989 ◽

Vol 9 (7) ◽

pp. 2906-2913 ◽

Cited By ~ 14

Author(s):

S C Francesconi ◽

S Eisenberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Binding ◽

Sodium Dodecyl ◽

Molecular Size ◽

Protein Band ◽

Binding Activity ◽

Physical Parameters ◽

Major Protein ◽

Dna Binding Activity ◽

Major Protein Band

We previously identified a protein activity from Saccharomyces cerevisiae, OBF1, that bound specifically to a DNA element present in autonomously replicating sequences ARS120 and ARS121 (S. Eisenberg C. Civalier, and B. K. Tye, Proc. Natl. Acad. Sci. USA 85:743-746, 1988). OBF1 has now been purified to near homogeneity by conventional protein and DNA affinity chromatography. Electrophoresis of the purified protein in sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two polypeptides. The major protein band had a relative molecular size of 123 kilodaltons, and the minor protein band, which constituted only a small fraction of total protein, had a molecular size of 127 kilodaltons. Both polypeptides cochromatographed with the specific ARS120 DNA-binding activity and formed a stable protein-DNA complex, isolatable by sedimentation through sucrose gradients. Using antibodies, we have shown that both polypeptides are associated with the isolated protein-DNA complexes. The ARS DNA-binding activity had a Stokes radius of 54 A (5.4 nm) and a sedimentation coefficient of 4.28S, as determined by gel filtration and sedimentation through glycerol gradients, respectively. These physical parameters, together with the denatured molecular size values, suggested that the proteins exist in solution as asymmetric monomers. Since both polypeptides recognized identical sequences and had similar physical properties, they are probably related. In addition to binding to ARS120, we found that purified OBF1 bounds with equal affinity to ARS121 and with 5- and 10-fold-lower affinity to ARS1 and HMRE, respectively. Furthermore, in the accompanying paper (S. S. Walker, S. C. Francesconi, B. K. Tye, and S. Eisenberg, Mol. Cell. Biol. 9:2914-2921, 1989), we demonstrate the existence of a high, direct correlation between the ability of purify OBF1 to bind to ARS121 and optimal in vivo ARS121 activity as an origin of replication. These findings, taken together, suggest a role for OBF1 in ARS function, presumably at the level of initiation of DNA replication at the ARS.

Download Full-text