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. 2017 Mar 18;6:e23474. doi: 10.7554/eLife.23474

Figure 5. DNA-binding of yCAF1 is required for DNA synthesis-coupled nucleosome assembly.

(A) Nucleosome assembly reactions with either non-UV-treated plasmid (pBS0) or plasmid irradiated with UV (pBSuv) in presence of [α−32P]. After an incubation time of 5 or 180 min, DNA was extracted, resolved on an agarose gel and visualized by ethidium bromide staining (EtBr) or by autoradiography (Autradiogram). (B) 150 ng of pBSuv plasmid was incubated with p150-depleted HSE extracts and complemented with the indicated amounts of yCAF1. After 3 hr incubation, DNA was extracted, resolved on and agarose gel and visualized as above. (C) As in (B) but reactions were complemented with 125 ng of the different yCAF1 variants and the amount of pBSuv plasmid was increased to 300 ng (D) As in (B) but reactions were complemented with 100 ng of the different yCAF1 variants. (I) Supercoiled and (II) relaxed plasmid. All reactions were repeated two times with consistency.

DOI: http://dx.doi.org/10.7554/eLife.23474.018

Figure 5.

Figure 5—figure supplement 1. Depletion of Xenopus p150 from HSE.

Figure 5—figure supplement 1.

(A) p150- or mock-depleted HSEs were analyzed by Western blotting using the indicated antibodies. (B) Nucleosome assembly reactions with 300 ng of pBSuv plasmid and 50 or 100 ng of yCAF1 variants showing that none of the yCAF1 mutants reach wild-type level activity. (C) Western blot of a FLAG pulldown of yeast PCNA with FLAG tagged yCAF1 WT or yCAF1V to investigate the effect of the N-terminal mutant on PCNA binding.
DOI: http://dx.doi.org/10.7554/eLife.23474.019