Structured Review

TaKaRa mnase
The proximal region of the DRAM promoter is sensitive to nuclease digestion. (A) Schematic representation of the primer sets used for CHART-PCR. (B) R6 region of the DRAM promoter is more sensitive to DNase I and <t>MNase</t> digestion. The genomic <t>DNA</t> was isolated from Hep3B and HepG2 cells and analyzed by quantitative PCR. The data was shown as percentage of digested DNAs to undigested DNAs. (C) Serum deprivation increases the accessibility of R5, R6 and R7 regions to nuclease digestion in Hep3B and HepG2 cells.
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Images

1) Product Images from "Serum Starvation Induces DRAM Expression in Liver Cancer Cells via Histone Modifications within Its Promoter Locus"

Article Title: Serum Starvation Induces DRAM Expression in Liver Cancer Cells via Histone Modifications within Its Promoter Locus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0050502

The proximal region of the DRAM promoter is sensitive to nuclease digestion. (A) Schematic representation of the primer sets used for CHART-PCR. (B) R6 region of the DRAM promoter is more sensitive to DNase I and MNase digestion. The genomic DNA was isolated from Hep3B and HepG2 cells and analyzed by quantitative PCR. The data was shown as percentage of digested DNAs to undigested DNAs. (C) Serum deprivation increases the accessibility of R5, R6 and R7 regions to nuclease digestion in Hep3B and HepG2 cells.
Figure Legend Snippet: The proximal region of the DRAM promoter is sensitive to nuclease digestion. (A) Schematic representation of the primer sets used for CHART-PCR. (B) R6 region of the DRAM promoter is more sensitive to DNase I and MNase digestion. The genomic DNA was isolated from Hep3B and HepG2 cells and analyzed by quantitative PCR. The data was shown as percentage of digested DNAs to undigested DNAs. (C) Serum deprivation increases the accessibility of R5, R6 and R7 regions to nuclease digestion in Hep3B and HepG2 cells.

Techniques Used: Polymerase Chain Reaction, Isolation, Real-time Polymerase Chain Reaction

2) Product Images from "MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast"

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky502

Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.
Figure Legend Snippet: Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.

Techniques Used:

MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.
Figure Legend Snippet: MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.

Techniques Used: Incubation, Atomic Absorption Spectroscopy

Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).
Figure Legend Snippet: Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).

Techniques Used: Sequencing

3) Product Images from "MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast"

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky502

Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.
Figure Legend Snippet: Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.

Techniques Used:

MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.
Figure Legend Snippet: MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.

Techniques Used: Incubation, Atomic Absorption Spectroscopy

Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).
Figure Legend Snippet: Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).

Techniques Used: Sequencing

Correlation between MNase digestions and contents of site-exposure sequence by comparing the two ends of a nucleosome. Correlations between MNase digestions and AA/TT contents in the 1- and 3-min assays are shown on the left and right panels of ( A ), respectively. Similarly, ( B ) shows the correlations of MNase digestion versus AAAA/TTTT content from the 1- and 3-min assays. The shaded rectangle regions in red indicate that the entry site of a nucleosome with more AA/TTs or AAAA/TTTTs gets more digested; the regions in blue indicate that the exit site with more AA/TTs or AAAA/TTTTs gets more digested. The 20 +1 nucleosomes are divided into two groups based on the numbers of s ite e xposure s equence e lements (SESEs, defined as discrete AAAA or TTTT segments) in their sequences. Specifically, nucleosomes with SESEs (coloured in black) are those with three or more SESEs on either strand of the nucleosomes, including nuc01, nuc02, nuc03, nuc05, nuc07, nuc10 and nuc20. Oppositely, nucleosomes with no or fewer SESEs (coloured in orange) consisting of the rest of the +1 nucleosomes, are those with two or fewer SESEs on each strand. Correlation coefficients for each class of nucleosomes under each incubation time are also indicated.
Figure Legend Snippet: Correlation between MNase digestions and contents of site-exposure sequence by comparing the two ends of a nucleosome. Correlations between MNase digestions and AA/TT contents in the 1- and 3-min assays are shown on the left and right panels of ( A ), respectively. Similarly, ( B ) shows the correlations of MNase digestion versus AAAA/TTTT content from the 1- and 3-min assays. The shaded rectangle regions in red indicate that the entry site of a nucleosome with more AA/TTs or AAAA/TTTTs gets more digested; the regions in blue indicate that the exit site with more AA/TTs or AAAA/TTTTs gets more digested. The 20 +1 nucleosomes are divided into two groups based on the numbers of s ite e xposure s equence e lements (SESEs, defined as discrete AAAA or TTTT segments) in their sequences. Specifically, nucleosomes with SESEs (coloured in black) are those with three or more SESEs on either strand of the nucleosomes, including nuc01, nuc02, nuc03, nuc05, nuc07, nuc10 and nuc20. Oppositely, nucleosomes with no or fewer SESEs (coloured in orange) consisting of the rest of the +1 nucleosomes, are those with two or fewer SESEs on each strand. Correlation coefficients for each class of nucleosomes under each incubation time are also indicated.

Techniques Used: Sequencing, Incubation

4) Product Images from "Crystal structure and stable property of the cancer-associated heterotypic nucleosome containing CENP-A and H3.3"

Article Title: Crystal structure and stable property of the cancer-associated heterotypic nucleosome containing CENP-A and H3.3

Journal: Scientific Reports

doi: 10.1038/srep07115

The DNA end close to CENP-A is asymmetrically flexible in the CENP-A/H3.3 nucleosome. (a) MNase assay. The H3.3, CENP-A, and CENP-A/H3.3 nucleosomes were treated with MNase (0, 0.3, 0.5 and 0.7 units), and the resulting DNA fragments were analyzed by native PAGE. The gel image shown is a representative of four independent experiments, in which similar results were obtained. (b) ExoIII assay. The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes were incubated with or without 2.0 units of ExoIII for 2.5, 5 and 7.5 minutes at 37°C, and the resultant DNA fragments were extracted and analyzed by 14% denaturing PAGE with 7 M urea. The gel image shown is a representative of three independent experiments, in which similar results were obtained.
Figure Legend Snippet: The DNA end close to CENP-A is asymmetrically flexible in the CENP-A/H3.3 nucleosome. (a) MNase assay. The H3.3, CENP-A, and CENP-A/H3.3 nucleosomes were treated with MNase (0, 0.3, 0.5 and 0.7 units), and the resulting DNA fragments were analyzed by native PAGE. The gel image shown is a representative of four independent experiments, in which similar results were obtained. (b) ExoIII assay. The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes were incubated with or without 2.0 units of ExoIII for 2.5, 5 and 7.5 minutes at 37°C, and the resultant DNA fragments were extracted and analyzed by 14% denaturing PAGE with 7 M urea. The gel image shown is a representative of three independent experiments, in which similar results were obtained.

Techniques Used: Clear Native PAGE, Incubation, Polyacrylamide Gel Electrophoresis

The DNA end close to CENP-A is asymmetrically flexible in the CENP-A/H3.3 nucleosome. (a) MNase assay. The H3.3, CENP-A, and CENP-A/H3.3 nucleosomes were treated with MNase (0, 0.3, 0.5 and 0.7 units), and the resulting DNA fragments were analyzed by native PAGE. The gel image shown is a representative of four independent experiments, in which similar results were obtained. (b) ExoIII assay. The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes were incubated with or without 2.0 units of ExoIII for 2.5, 5 and 7.5 minutes at 37°C, and the resultant DNA fragments were extracted and analyzed by 14% denaturing PAGE with 7 M urea. The gel image shown is a representative of three independent experiments, in which similar results were obtained.
Figure Legend Snippet: The DNA end close to CENP-A is asymmetrically flexible in the CENP-A/H3.3 nucleosome. (a) MNase assay. The H3.3, CENP-A, and CENP-A/H3.3 nucleosomes were treated with MNase (0, 0.3, 0.5 and 0.7 units), and the resulting DNA fragments were analyzed by native PAGE. The gel image shown is a representative of four independent experiments, in which similar results were obtained. (b) ExoIII assay. The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes were incubated with or without 2.0 units of ExoIII for 2.5, 5 and 7.5 minutes at 37°C, and the resultant DNA fragments were extracted and analyzed by 14% denaturing PAGE with 7 M urea. The gel image shown is a representative of three independent experiments, in which similar results were obtained.

Techniques Used: Clear Native PAGE, Incubation, Polyacrylamide Gel Electrophoresis

5) Product Images from "MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast"

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky502

Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.
Figure Legend Snippet: Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.

Techniques Used:

MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.
Figure Legend Snippet: MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.

Techniques Used: Incubation, Atomic Absorption Spectroscopy

Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).
Figure Legend Snippet: Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).

Techniques Used: Sequencing

Correlation between MNase digestions and contents of site-exposure sequence by comparing the two ends of a nucleosome. Correlations between MNase digestions and AA/TT contents in the 1- and 3-min assays are shown on the left and right panels of ( A ), respectively. Similarly, ( B ) shows the correlations of MNase digestion versus AAAA/TTTT content from the 1- and 3-min assays. The shaded rectangle regions in red indicate that the entry site of a nucleosome with more AA/TTs or AAAA/TTTTs gets more digested; the regions in blue indicate that the exit site with more AA/TTs or AAAA/TTTTs gets more digested. The 20 +1 nucleosomes are divided into two groups based on the numbers of s ite e xposure s equence e lements (SESEs, defined as discrete AAAA or TTTT segments) in their sequences. Specifically, nucleosomes with SESEs (coloured in black) are those with three or more SESEs on either strand of the nucleosomes, including nuc01, nuc02, nuc03, nuc05, nuc07, nuc10 and nuc20. Oppositely, nucleosomes with no or fewer SESEs (coloured in orange) consisting of the rest of the +1 nucleosomes, are those with two or fewer SESEs on each strand. Correlation coefficients for each class of nucleosomes under each incubation time are also indicated.
Figure Legend Snippet: Correlation between MNase digestions and contents of site-exposure sequence by comparing the two ends of a nucleosome. Correlations between MNase digestions and AA/TT contents in the 1- and 3-min assays are shown on the left and right panels of ( A ), respectively. Similarly, ( B ) shows the correlations of MNase digestion versus AAAA/TTTT content from the 1- and 3-min assays. The shaded rectangle regions in red indicate that the entry site of a nucleosome with more AA/TTs or AAAA/TTTTs gets more digested; the regions in blue indicate that the exit site with more AA/TTs or AAAA/TTTTs gets more digested. The 20 +1 nucleosomes are divided into two groups based on the numbers of s ite e xposure s equence e lements (SESEs, defined as discrete AAAA or TTTT segments) in their sequences. Specifically, nucleosomes with SESEs (coloured in black) are those with three or more SESEs on either strand of the nucleosomes, including nuc01, nuc02, nuc03, nuc05, nuc07, nuc10 and nuc20. Oppositely, nucleosomes with no or fewer SESEs (coloured in orange) consisting of the rest of the +1 nucleosomes, are those with two or fewer SESEs on each strand. Correlation coefficients for each class of nucleosomes under each incubation time are also indicated.

Techniques Used: Sequencing, Incubation

6) Product Images from "MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast"

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky502

Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.
Figure Legend Snippet: Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.

Techniques Used:

MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.
Figure Legend Snippet: MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.

Techniques Used: Incubation, Atomic Absorption Spectroscopy

Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).
Figure Legend Snippet: Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).

Techniques Used: Sequencing

7) Product Images from "MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast"

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky502

Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.
Figure Legend Snippet: Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.

Techniques Used:

MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.
Figure Legend Snippet: MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.

Techniques Used: Incubation, Atomic Absorption Spectroscopy

Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).
Figure Legend Snippet: Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).

Techniques Used: Sequencing

8) Product Images from "Crystal structure and stable property of the cancer-associated heterotypic nucleosome containing CENP-A and H3.3"

Article Title: Crystal structure and stable property of the cancer-associated heterotypic nucleosome containing CENP-A and H3.3

Journal: Scientific Reports

doi: 10.1038/srep07115

The DNA end close to CENP-A is asymmetrically flexible in the CENP-A/H3.3 nucleosome. (a) MNase assay. The H3.3, CENP-A, and CENP-A/H3.3 nucleosomes were treated with MNase (0, 0.3, 0.5 and 0.7 units), and the resulting DNA fragments were analyzed by native PAGE. The gel image shown is a representative of four independent experiments, in which similar results were obtained. (b) ExoIII assay. The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes were incubated with or without 2.0 units of ExoIII for 2.5, 5 and 7.5 minutes at 37°C, and the resultant DNA fragments were extracted and analyzed by 14% denaturing PAGE with 7 M urea. The gel image shown is a representative of three independent experiments, in which similar results were obtained.
Figure Legend Snippet: The DNA end close to CENP-A is asymmetrically flexible in the CENP-A/H3.3 nucleosome. (a) MNase assay. The H3.3, CENP-A, and CENP-A/H3.3 nucleosomes were treated with MNase (0, 0.3, 0.5 and 0.7 units), and the resulting DNA fragments were analyzed by native PAGE. The gel image shown is a representative of four independent experiments, in which similar results were obtained. (b) ExoIII assay. The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes were incubated with or without 2.0 units of ExoIII for 2.5, 5 and 7.5 minutes at 37°C, and the resultant DNA fragments were extracted and analyzed by 14% denaturing PAGE with 7 M urea. The gel image shown is a representative of three independent experiments, in which similar results were obtained.

Techniques Used: Clear Native PAGE, Incubation, Polyacrylamide Gel Electrophoresis

The DNA end close to CENP-A is asymmetrically flexible in the CENP-A/H3.3 nucleosome. (a) MNase assay. The H3.3, CENP-A, and CENP-A/H3.3 nucleosomes were treated with MNase (0, 0.3, 0.5 and 0.7 units), and the resulting DNA fragments were analyzed by native PAGE. The gel image shown is a representative of four independent experiments, in which similar results were obtained. (b) ExoIII assay. The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes were incubated with or without 2.0 units of ExoIII for 2.5, 5 and 7.5 minutes at 37°C, and the resultant DNA fragments were extracted and analyzed by 14% denaturing PAGE with 7 M urea. The gel image shown is a representative of three independent experiments, in which similar results were obtained.
Figure Legend Snippet: The DNA end close to CENP-A is asymmetrically flexible in the CENP-A/H3.3 nucleosome. (a) MNase assay. The H3.3, CENP-A, and CENP-A/H3.3 nucleosomes were treated with MNase (0, 0.3, 0.5 and 0.7 units), and the resulting DNA fragments were analyzed by native PAGE. The gel image shown is a representative of four independent experiments, in which similar results were obtained. (b) ExoIII assay. The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes were incubated with or without 2.0 units of ExoIII for 2.5, 5 and 7.5 minutes at 37°C, and the resultant DNA fragments were extracted and analyzed by 14% denaturing PAGE with 7 M urea. The gel image shown is a representative of three independent experiments, in which similar results were obtained.

Techniques Used: Clear Native PAGE, Incubation, Polyacrylamide Gel Electrophoresis

9) Product Images from "MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast"

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky502

Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.
Figure Legend Snippet: Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.

Techniques Used:

MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.
Figure Legend Snippet: MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.

Techniques Used: Incubation, Atomic Absorption Spectroscopy

Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).
Figure Legend Snippet: Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).

Techniques Used: Sequencing

Related Articles

Incubation:

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast
Article Snippet: .. The nucleosome mixture (94 nM), containing all types of nucleosomes, was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1, 3, 6 and 10 min, in 50 mM Tris–HCl (pH 8.0) buffer, containing 2.5 mM CaCl2 , 69 mM NaCl, 81 mM KCl and 1.9 mM dithiothreitol. .. The reaction was stopped by adding half volume of deproteinization solution (20 mM Tris–HCl (pH 8.0), 20 mM EDTA, 0.5 mg/ml proteinase K (Roche) and 0.1% sodium dodecyl sulphate).

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast
Article Snippet: .. For the MNase digestion experiments with nuc19 and its mutants , each nucleosome (94 nM) was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as described above. ..

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast
Article Snippet: .. MNase digestion assays The nucleosome mixture (94 nM), containing all types of nucleosomes, was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1, 3, 6 and 10 min, in 50 mM Tris–HCl (pH 8.0) buffer, containing 2.5 mM CaCl2 , 69 mM NaCl, 81 mM KCl and 1.9 mM dithiothreitol. .. The reaction was stopped by adding half volume of deproteinization solution (20 mM Tris–HCl (pH 8.0), 20 mM EDTA, 0.5 mg/ml proteinase K (Roche) and 0.1% sodium dodecyl sulphate).

Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast
Article Snippet: .. For the MNase digestion experiments with free DNAs, DNA mixture (94 nM) was incubated with 0.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as nucleosome digestion experiments. .. The libraries were prepared by using the NEBNext Ultra DNA Library Prep Kit (New England Biolabs).

other:

Article Title: Serum Starvation Induces DRAM Expression in Liver Cancer Cells via Histone Modifications within Its Promoter Locus
Article Snippet: Chromatin accessibility analysis Accessibility of DNA to DNase I digestion and MNase (Takara, Inc., Dalian, China) were analyzed using chromatin accessibility through CHART-PCR as described previously – .

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  • 93
    TaKaRa mnase digestion
    Frequencies of occurrence of <t>DNA</t> dinucleotide steps in the +1 nucleosomes of yeast and the sketch of <t>MNase-seq</t> experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.
    Mnase Digestion, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mnase digestion/product/TaKaRa
    Average 93 stars, based on 42 article reviews
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    mnase digestion - by Bioz Stars, 2020-05
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    95
    TaKaRa micrococcal nuclease mnase
    Chromatin structure around ade6-3049 is more open than around ade6-3057 , and HRA is involved in the chromatin regulation. (A) <t>MNase</t> sensitivity of chromatin around ade6-3049/3057 . Indicated cells of the pat1-114 background were induced to enter meiosis by temperature-shift method, and harvested 3 hr after the induction. MNase-digested <t>DNA</t> was prepared by MNase (0, 20, or 30 units) treatment of chromatin fraction and subsequent purification. DNA was further cut with Sac I, fractionated on 1.2% agaraose gel, and analyzed by Southern blotting using the probe indicated by the open box. The vertical arrow and the open arrowhead show the ade6 ORF and the 3049/3057 site, respectively. The numbers on the left and the right side of the panel indicate the positions of λ/ Eco T14 I fragments used for electrophoresis marker and the positions from the first A of the ade6 ORF, respectively. The dotted lines indicate a region in which MNase-sensitive sites are clustered around ade6-3049 . Presented is an example from two independent experiments, whose results were similar to each other. (B) HRA deletion reduced acetylation level of H3K9 around ade6-3049 . Indicated cells of the pat1-114 ). DNA isolated from immunoprecipitates and whole-cell extracts was analyzed by real-time qPCR, where fragments corresponding to ade6-3049/3057 and the prp3 + promoter were amplified. Relative enrichment at ade6-3049/3057 over prp3 is shown. Bar graphs are created based on mean values of two independent experiments (shown by ○).
    Micrococcal Nuclease Mnase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/micrococcal nuclease mnase/product/TaKaRa
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    Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.

    Journal: Nucleic Acids Research

    Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

    doi: 10.1093/nar/gky502

    Figure Lengend Snippet: Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.

    Article Snippet: For the MNase digestion experiments with free DNAs, DNA mixture (94 nM) was incubated with 0.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as nucleosome digestion experiments.

    Techniques:

    MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.

    Journal: Nucleic Acids Research

    Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

    doi: 10.1093/nar/gky502

    Figure Lengend Snippet: MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.

    Article Snippet: For the MNase digestion experiments with free DNAs, DNA mixture (94 nM) was incubated with 0.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as nucleosome digestion experiments.

    Techniques: Incubation, Atomic Absorption Spectroscopy

    Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).

    Journal: Nucleic Acids Research

    Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

    doi: 10.1093/nar/gky502

    Figure Lengend Snippet: Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).

    Article Snippet: For the MNase digestion experiments with free DNAs, DNA mixture (94 nM) was incubated with 0.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as nucleosome digestion experiments.

    Techniques: Sequencing

    The proximal region of the DRAM promoter is sensitive to nuclease digestion. (A) Schematic representation of the primer sets used for CHART-PCR. (B) R6 region of the DRAM promoter is more sensitive to DNase I and MNase digestion. The genomic DNA was isolated from Hep3B and HepG2 cells and analyzed by quantitative PCR. The data was shown as percentage of digested DNAs to undigested DNAs. (C) Serum deprivation increases the accessibility of R5, R6 and R7 regions to nuclease digestion in Hep3B and HepG2 cells.

    Journal: PLoS ONE

    Article Title: Serum Starvation Induces DRAM Expression in Liver Cancer Cells via Histone Modifications within Its Promoter Locus

    doi: 10.1371/journal.pone.0050502

    Figure Lengend Snippet: The proximal region of the DRAM promoter is sensitive to nuclease digestion. (A) Schematic representation of the primer sets used for CHART-PCR. (B) R6 region of the DRAM promoter is more sensitive to DNase I and MNase digestion. The genomic DNA was isolated from Hep3B and HepG2 cells and analyzed by quantitative PCR. The data was shown as percentage of digested DNAs to undigested DNAs. (C) Serum deprivation increases the accessibility of R5, R6 and R7 regions to nuclease digestion in Hep3B and HepG2 cells.

    Article Snippet: Chromatin accessibility analysis Accessibility of DNA to DNase I digestion and MNase (Takara, Inc., Dalian, China) were analyzed using chromatin accessibility through CHART-PCR as described previously – .

    Techniques: Polymerase Chain Reaction, Isolation, Real-time Polymerase Chain Reaction

    Correlation between MNase digestions and contents of site-exposure sequence by comparing the two ends of a nucleosome. Correlations between MNase digestions and AA/TT contents in the 1- and 3-min assays are shown on the left and right panels of ( A ), respectively. Similarly, ( B ) shows the correlations of MNase digestion versus AAAA/TTTT content from the 1- and 3-min assays. The shaded rectangle regions in red indicate that the entry site of a nucleosome with more AA/TTs or AAAA/TTTTs gets more digested; the regions in blue indicate that the exit site with more AA/TTs or AAAA/TTTTs gets more digested. The 20 +1 nucleosomes are divided into two groups based on the numbers of s ite e xposure s equence e lements (SESEs, defined as discrete AAAA or TTTT segments) in their sequences. Specifically, nucleosomes with SESEs (coloured in black) are those with three or more SESEs on either strand of the nucleosomes, including nuc01, nuc02, nuc03, nuc05, nuc07, nuc10 and nuc20. Oppositely, nucleosomes with no or fewer SESEs (coloured in orange) consisting of the rest of the +1 nucleosomes, are those with two or fewer SESEs on each strand. Correlation coefficients for each class of nucleosomes under each incubation time are also indicated.

    Journal: Nucleic Acids Research

    Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

    doi: 10.1093/nar/gky502

    Figure Lengend Snippet: Correlation between MNase digestions and contents of site-exposure sequence by comparing the two ends of a nucleosome. Correlations between MNase digestions and AA/TT contents in the 1- and 3-min assays are shown on the left and right panels of ( A ), respectively. Similarly, ( B ) shows the correlations of MNase digestion versus AAAA/TTTT content from the 1- and 3-min assays. The shaded rectangle regions in red indicate that the entry site of a nucleosome with more AA/TTs or AAAA/TTTTs gets more digested; the regions in blue indicate that the exit site with more AA/TTs or AAAA/TTTTs gets more digested. The 20 +1 nucleosomes are divided into two groups based on the numbers of s ite e xposure s equence e lements (SESEs, defined as discrete AAAA or TTTT segments) in their sequences. Specifically, nucleosomes with SESEs (coloured in black) are those with three or more SESEs on either strand of the nucleosomes, including nuc01, nuc02, nuc03, nuc05, nuc07, nuc10 and nuc20. Oppositely, nucleosomes with no or fewer SESEs (coloured in orange) consisting of the rest of the +1 nucleosomes, are those with two or fewer SESEs on each strand. Correlation coefficients for each class of nucleosomes under each incubation time are also indicated.

    Article Snippet: MNase digestion assays The nucleosome mixture (94 nM), containing all types of nucleosomes, was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1, 3, 6 and 10 min, in 50 mM Tris–HCl (pH 8.0) buffer, containing 2.5 mM CaCl2 , 69 mM NaCl, 81 mM KCl and 1.9 mM dithiothreitol.

    Techniques: Sequencing, Incubation

    Chromatin structure around ade6-3049 is more open than around ade6-3057 , and HRA is involved in the chromatin regulation. (A) MNase sensitivity of chromatin around ade6-3049/3057 . Indicated cells of the pat1-114 background were induced to enter meiosis by temperature-shift method, and harvested 3 hr after the induction. MNase-digested DNA was prepared by MNase (0, 20, or 30 units) treatment of chromatin fraction and subsequent purification. DNA was further cut with Sac I, fractionated on 1.2% agaraose gel, and analyzed by Southern blotting using the probe indicated by the open box. The vertical arrow and the open arrowhead show the ade6 ORF and the 3049/3057 site, respectively. The numbers on the left and the right side of the panel indicate the positions of λ/ Eco T14 I fragments used for electrophoresis marker and the positions from the first A of the ade6 ORF, respectively. The dotted lines indicate a region in which MNase-sensitive sites are clustered around ade6-3049 . Presented is an example from two independent experiments, whose results were similar to each other. (B) HRA deletion reduced acetylation level of H3K9 around ade6-3049 . Indicated cells of the pat1-114 ). DNA isolated from immunoprecipitates and whole-cell extracts was analyzed by real-time qPCR, where fragments corresponding to ade6-3049/3057 and the prp3 + promoter were amplified. Relative enrichment at ade6-3049/3057 over prp3 is shown. Bar graphs are created based on mean values of two independent experiments (shown by ○).

    Journal: Genetics

    Article Title: Correlation of Meiotic DSB Formation and Transcription Initiation Around Fission Yeast Recombination Hotspots

    doi: 10.1534/genetics.116.197954

    Figure Lengend Snippet: Chromatin structure around ade6-3049 is more open than around ade6-3057 , and HRA is involved in the chromatin regulation. (A) MNase sensitivity of chromatin around ade6-3049/3057 . Indicated cells of the pat1-114 background were induced to enter meiosis by temperature-shift method, and harvested 3 hr after the induction. MNase-digested DNA was prepared by MNase (0, 20, or 30 units) treatment of chromatin fraction and subsequent purification. DNA was further cut with Sac I, fractionated on 1.2% agaraose gel, and analyzed by Southern blotting using the probe indicated by the open box. The vertical arrow and the open arrowhead show the ade6 ORF and the 3049/3057 site, respectively. The numbers on the left and the right side of the panel indicate the positions of λ/ Eco T14 I fragments used for electrophoresis marker and the positions from the first A of the ade6 ORF, respectively. The dotted lines indicate a region in which MNase-sensitive sites are clustered around ade6-3049 . Presented is an example from two independent experiments, whose results were similar to each other. (B) HRA deletion reduced acetylation level of H3K9 around ade6-3049 . Indicated cells of the pat1-114 ). DNA isolated from immunoprecipitates and whole-cell extracts was analyzed by real-time qPCR, where fragments corresponding to ade6-3049/3057 and the prp3 + promoter were amplified. Relative enrichment at ade6-3049/3057 over prp3 is shown. Bar graphs are created based on mean values of two independent experiments (shown by ○).

    Article Snippet: Genomic DNA was treated with 0, 20, or 30 units of micrococcal nuclease (MNase) (Takara), digested with Sac I, and separated on a 1.2% agarose gel.

    Techniques: Purification, Southern Blot, Electrophoresis, Marker, Isolation, Real-time Polymerase Chain Reaction, Amplification