PBSK-Zeo backbone region 2 (B). Restriction inhibition assay demonstrates that the two
PBSK-Zeo backbone region 2 (B). Restriction inhibition assay demonstrates that the two regions are not 4-Hydroxytamoxifen cancer equally accessible since region 1 is highly protected when chromatinized whereas region 2 remains highly accessible for restriction site cutting even in the nucleosomal structure (see Additional file 1: Figure S1).U3 and U5 were used in concerted integration assays performed using naked or chromatinized p5S acceptor. As reported in Figure 2A and B, under these conditions all the enzymes were found highly active using their specific donor DNA and the naked p5S receptor plasmid. Specific isolation and quantification of the physiological FSI integration products (Figure 2C) show that the enzymes were equally active leading to a similar amount of integrants (200 to 225 per experiment). However, despite their similar activity on naked DNA, large differences were found in their activity on nucleosomal templates. Indeed, in contrast to HIV-1, the activity of PFV and MLV INs were strongly stimulated on chromatinized receptor plasmid leading to a 4- to 5-fold increase in the integration products (Figure 2A as well as quantification in 2B and C). No significant change in integration fidelity was found using the chromatinized vector comparing to the naked one (Additional file 3: Figure S3).To confirm that different retroviral INs could be affected differently by chromatin in vitro?ASV enzyme was tested under its previously reported optimal conditions [52]. Under these conditions, ASV IN was found to be more active on naked DNA than HIV-1, PFV and MLV enzymes PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27693494 catalyzing the formation of a greater amount of integration products (Figure 2A and quantification in 2B). Cloning and quantification of the circular FSI forms, the most representative for the physiological integration reaction observed in cells, confirmed that ASV IN was more active in vitro (Figure 2C) and preferentially displayed the expected 6 bp target site duplication (Additional file 3: Figure S3). However, despite its higher activity found on naked DNA, ASV integration was strongly inhibited by nucleosomes assembly as observed for HIV-1 IN (see Figure 2A and quantification in 2B). Additionally, specific quantification of the circular FSI products also revealed a strong decrease in the number of integrants on nucleosomalBenleulmi et al. Retrovirology (2015) 12:Page 5 ofFigure PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28381880 2 HIV-1, PFV, MLV and ASV in vitro integration on naked and chromatinized p5S vectors. Concerted integration assay was performed with 10 ng of donor DNA and 100 ng of p5S naked plasmids (lanes 1), or polynucleosomal vectors assembled with increasing amounts of histones expressed as DNA/histones mass ratio (g/g) (1/1.1, lanes 2; 1/1.3, lanes 3) and either HIV-1, PFV, MLV (100nM) or ASV (600 nM) integrases. The reaction products were loaded onto 1 agarose gel and a representative set of experiments is shown in the figure (A). The position and structures of the donor substrate and different products obtained after half-site (HSI), full-site (FSI) and donor/donor integration (d/d) are shown. The circular FSI + HSI and linear FSI products were quantified on gel using the ImageJ software and are shown respectively in the left and right panels in (B). The circular FSI products were specifically quantified by cloning in bacteria and shown as the number of ampicillin-, kanamycin- and tetracycline-resistant selected clones as a percentage of integration reaction control performed with naked vectors (C). All the values are.

Leave a Reply