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. 2017 Mar 21;6:e22512. doi: 10.7554/eLife.22512

Figure 1. Mcm2-7 helicase loading onto nucleosomal DNA templates.

(A) Nucleosomes were remodeled with bead-coupled ARS1-containing linear DNA, ISW1a, yeast histone octamers and Nap1. Nucleosome assembly was assessed after partial MNase digestion. (B) Outline of the helicase-loading assay using nucleosomal DNA. (C). Comparison of helicase loading on naked DNA and on ISW1a-remodeled nucleosomal DNA. DNA templates were washed with high-salt (H) or low-salt (L) buffer after loading. Template-associated Mcm2-7, ORC and H2B was detected by immunoblot. (D) Helicase loading onto either wild-type (WT) or A-B2- (mut) (Heller et al., 2011) ARS1-containing DNA. As indicated, nucleosomal DNA was remodeled with ISW1a. Assays were performed in either 125 mM (to allow increased origin non-specific helicase loading) or 300 mM (origin specific helicase loading) potassium glutamate. After a high salt wash, DNA-associated Mcm2-7 was detected by immunoblot.

DOI: http://dx.doi.org/10.7554/eLife.22512.003

Figure 1.

Figure 1—figure supplement 1. Purified proteins used in the in vitro nucleosome.

Figure 1—figure supplement 1.

assembly reactions. (A) Purified yeast histone octamers and Nap1 were separated by. SDS-AGE and visualized by Coomassie staining. (B) Purified CREs were separated. by SDS-PAGE and visualized by Coomassie staining.
DOI: http://dx.doi.org/10.7554/eLife.22512.004
Figure 1—figure supplement 2. Preparation of in vitro nucleosome templates.

Figure 1—figure supplement 2.

(A) Nucleosomes were assembled with increasing amounts of histone octamers and fixed amount of DNA with ISW1a. Nucleosome assembly was as in Figure 1A. (B) Removal of CRE and unassociated proteins from in- vitro assembled nucleosomes. Amount of CRE and Nap1 associated with nucleosomal DNA before and after washing was detected by anti-CBP (Ioc3-TAP, Ioc2-TAP, Ino80-TAP, Chd1-TAP, Swi2-TAP and Rsc2-TAP), anti-FLAG (Isw2-FLAG), anti-H2B and anti-6xHis (Nap1) immunoblots.
DOI: http://dx.doi.org/10.7554/eLife.22512.005
Figure 1—figure supplement 3. The ATPase activities of in vitro purified chromatin remodeling enzymes.

Figure 1—figure supplement 3.

The ATPase activities of chromatin remodeling enzymes were measured in the presence of 0.1 mg/ml plasmid DNA. The fractions of hydrolyzed ATP were normalized with 1 nM remodeling enzymes.
DOI: http://dx.doi.org/10.7554/eLife.22512.006