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Fig. 2 | Retrovirology

Fig. 2

From: Modulation of chromatin structure by the FACT histone chaperone complex regulates HIV-1 integration

Fig. 2

Effect of FACT complex on in vitro HIV-1 integration. A concerted integration assay was performed using 200 nM of HIV-1 IN, 10 ng of donor DNA, 50 ng of p5S naked or chromatinized plasmid DNA (PN DNA), and increasing concentrations of FACT complex. The reaction products were loaded onto 1% agarose gels, and a representative set of experiments performed with the wild-type integrase is reported in (a). The positions and structures of the donor substrate and the different half-site (HSI), full-site (FSI) and donor/donor integration (d/d) products are shown. Quantification of the total integration products (FSI, HSI and donor/donor) was performed via gel detection and is reported in (b) as the percentage relative to activity without FACT. Effect of FACT complex or SSRP1 or Spt16 proteins [optimal FACT concentration as determined in (b)] on integration into PN DNA was analyzed and integration efficiency was reported in (c). Effect of FACT nucleosome remodeling activity on integration catalyzed by HIV-1 IN on chromatinized substrates was analyzed by comparing chromatinized p5S vector (histone/DNA ratio = 1) treated with UV or untreated as reported in “Methods” section. The treated or untreated substrates were then used in concerted integration assays without FACT or with increasing concentrations of the complex. Quantification of the total integration products detected on gel (see representative experiment in Additional file 4: Figure S4) (FSI, HSI and donor/donor) was performed via gel detection and is reported in (d) as the percentage relative to activity detected on naked DNA (pre-normalized data obtained in the control experiments using naked DNA and without FACT are also reported as the percentage of integrated substrate). All values are shown as the mean ± standard deviation (error bars) of at least three independent sets of experiments

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