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. 2021 Oct 11;49(19):11241–11256. doi: 10.1093/nar/gkab886

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© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 5. — Effect of LEDGF/P75 on in vitro integration catalyzed by HIV-1 IN onto nucleosomes. Integration was performed using radiolabelled viral DNA, HIV-1 IN with or without LEDGF/p75 using either biotinylated naked 601 DNA or MN assembled on 601 sequence. The integration was quantified by measuring the remaining radioactivity on streptavidin beads after hammering the target DNA (A) or by loading the integration products on polyacrylamid 6–12% gradient gel (B). Data are reported in (A) as stimulation effect of LEDGF/p75 according to the control experiment performed without the cellular factor on either naked of MN targets. Data were obtained from at least three independent experiments and are reported as means ± SD. Statistics were performed by student test and the calculated p is reported on the figure. Integration product structures were analyzed on native polyacrylamide 6–12% gel as reported in (B). The half site integration (HSI) and full site integration (FSI) products are schematized in the figure. The positions of the integration sites on the nucleosomal DNA in the absence or in the presence of LEDGF/p75 were compared by scanning the gel migration profile using ImageJ software and reporting the radioactivity signal intensity in function of the migration distance from the top of the gel (representative analysis reported in the figure). Integration performed in the presence of LEDGF/p75 with MNs carrying histone modifications H3K36me2/me3 (C), tailless MNs (D) and tri-nucleosome (E) were performed under similar reaction conditions and products were quantified as described previously. Data were obtained from three to four independent experiments and are reported as means ± SD.