Ence of detergent, use of donor DNA containing various viral U
Ence of detergent, use of donor DNA containing various viral U3/U5 ends combination, see Additional file 4: Figure S4). Even if we can not completely rule out any bias link to the reaction conditions selected for the study (presence of PEG for example) the focus of the analysis on the full site integration products is expected to limit this bias. Indeed, even if the formation of a catalytically proficient intasome remains a limiting step with regard to integration efficacy, the reaction conditions should only affect the amount of functional intasomes formed and not the choice of the integration site dictated both the architecture of the intasome and the local PD173074MedChemExpress PD173074 target DNA structure. This is supported by the fact that the differences in the sensitivity toward nucleosomal density were found independent on the efficiency of concerted integration. Indeed, ASV was found more active than PFV on naked DNA but was also found inhibited by stable chromatin as HIV-1 (less active than PFV in catalyzing concerted integration events). Furthermore, the differences found between HIV-1/ASV and PFV/MLV INs were not dependent on the presence of the additional NED domain in PFV, indicating that the differential effect of chromatin on these enzymes in vitro is probably mainly due to local differences in the architecture of the catalytic pocket within the functional intasomes and not to global structural differences between the complexes. This is supported by the differences found between the HIV-1 integration reactions, leading to different staggered cuts in the target DNA. Indeed we previously showed that full site and half site integration could be impacted differently by nucleosome assembly in vitro [36]. Interestingly, differences were also found in this work regarding the effect of nucleosomes on the selectivity of HIV-1 integration reactions leading to 4 bp, 5 bp or 6 bp target DNA duplications (see Additional file 6: Figure S6). Indeed, while 5 bp and 6 bp integration reactions were highly disfavored in the stable chromatin region of the acceptor plasmid, 4 bp events were more widespread in the backbone with a clear preference for the high nucleosome density region. Since the ratio of chromatin assembly did not influence the proportion of these “non-physiological” integration events (Additional file 3: Figure S3), the latter are most likely catalyzed by aberrant intasomes structures as previously demonstrated [14]. Consequently, the most reasonable hypothesis that could account for their enrichment in nucleosome dense regions would be that the IN sensitivity toward chromatin is mainly driven by the structure of the IN/ viral DNA complex that can or cannot accommodate nucleosomal DNA depending on the relative position of the active sites. Because no bias in the PFV integration site positions was found in the naked version of the acceptor plasmid (Figure 5 and Additional file 5: Figure S5), our dataBenleulmi et al. Retrovirology (2015) 12:Page 12 ofindicate that the PFV and MLV intasomes can fit with compacted chromatin, in contrast to HIV-1 and ASV INs. The structure of the target DNA, and especially its bending, in the retroviral PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/29072704 intasome is expected to be governed by the space between the two catalytic PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28914615 sites involved in the staggered cut leading to different target DNA duplication size [8-10]. This target DNA curvature varying in the different intasomes could, thus, impact the nucleosome sensitivity of the retroviral enzymes. This is supported by the.

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