Skip to main content
. 2017 Mar 21;6:e22512. doi: 10.7554/eLife.22512

Figure 2. Comparison of helicase loading onto nucleosomal DNA templates remodeled with different CREs.

(A) Comparison of nucleosome assembly with different CREs. Nucleosomes were remodeled with the indicated CRE and assayed by partial MNase digestion. (B) Helicase loading onto nucleosomes remodeled with different CREs. After helicase loading, DNA was washed either with high-salt (H) or low-salt (L) buffer. Mcm2-7 and H2B DNA association was detected by immunoblot. (C) Comparison of origin-proximal nucleosome positioning established by different CREs. The positions of nucleosome dyads remodeled with the indicated CRE were analyzed by high-throughput MNase-Seq. Nucleosome dyad density (Y-axis) and the corresponding position of the dyad (X-axis) are plotted. Zero on the X-axis indicates the first nucleotide of the ARS1 consensus sequence (ACS). The elements of ARS1 (Marahrens and Stillman, 1992) are indicated above. (D) ORC association with nucleosomal DNA remodeled with different CREs. Template association of ORC was detected by immunoblot. (E) Addition of ORC during nucleosome assembly restores helicase loading on ISW2 and Chd1 templates. Nucleosomes were assembled onto ARS1 DNA with the indicated CRE in the presence or absence of ORC. Helicase loading was performed and analyzed as described in (B).

DOI: http://dx.doi.org/10.7554/eLife.22512.007

Figure 2.

Figure 2—figure supplement 1. Nucleosome assembly with different CREs and their ability to load Mcm2-7 helicase.

Figure 2—figure supplement 1.

(A) Histone H2B and H3 associated with nucleosomes assembled with the indicated CREs was detected by anti-H2B and H3 immunoblot. (B) Mono-nucleosomes produced from nucleosomal templates assembled with different CREs Similar amounts of ISW1a-, INO80-C, Chd1-, RSC- and SWI/SNF assembled templates were digested extensively with MNase and purified mononucleosomal DNA was analyzed by agarose gel electrophoresis. (C) Quantification of relative Mcm2-7 loading for nucleosomal templates assembled with the indicated CRE. The amount of Mcm2-7 quantified using online software ImajeJ and statistical analysis was performed using Prism software. For each assay three (n = 3) biological replicates were quantified. Mean value for ISW1a (High salt wash) reactions was calculated and set as the 100%. All the other values were calculated as a percentage of that mean value. Error bars indicate standard deviation (SD).
DOI: http://dx.doi.org/10.7554/eLife.22512.008
Figure 2—figure supplement 1—source data 1. Raw values used in the quantification of Figure 2B, left panel (n = 3).
DOI: http://dx.doi.org/10.7554/eLife.22512.009
elife-22512-fig2-figsupp1-data1.xlsx (27.6KB, xlsx)
DOI: 10.7554/eLife.22512.009
Figure 2—figure supplement 1—source data 2. Raw values used in the quantification of Figure 2B, right panel (n = 3).
DOI: http://dx.doi.org/10.7554/eLife.22512.010
elife-22512-fig2-figsupp1-data2.xlsx (31.6KB, xlsx)
DOI: 10.7554/eLife.22512.010
Figure 2—figure supplement 2. ORC1 BAH domain and Abf1 is dispensable for helicase loading of nucleosomal templates.

Figure 2—figure supplement 2.

(A) Purified ORC and ORCΔBAH were separated by SDS-PAGE and visualized by Coomassie staining. (B) Comparison of WT. and ΔBAH ORC mediated helicase loading on ISW1a-remodeled DNA templates. DNA templates were washed with high-salt (H) or low-salt (L) buffer after loading. Template associated Mcm2-7 and ORC was detected by immunoblot. (C) Helicase loading onto ISW1a, ISW2 and INO80-C templates were carried out with WT or ΔBAHORC. DNA templates were washed with high-salt (H) after loading. Template-associated Mcm2-7 and H2B was detected by immunoblot. (D) Purified Abf1 was separated by SDS-PAGE and visualized by Coomassie staining. (E) Comparison of Mcm2-7 loading for naked DNA and ISW1aremodeled nucleosomal templates. After helicase loading, DNA was washed with high salt. Mcm2-7 and H2B DNA association was detected by immunoblot. (F) Addition of Abf1 during nucleosome assembly do not restore helicase loading defects of ISW2 templates. Nucleosomes were remodeled onto ARS1 DNA with the indicated CRE in the presence/absence of ORC or Abf1 and ability load helicase was determined. Template associated Mcm2-7, Abf1(anti-Flag) and H2B was detected by immunoblot after high salt wash.
DOI: http://dx.doi.org/10.7554/eLife.22512.011