Monitoring the HIV-1 integrase enzymatic activity using atomic force microscopy in a 2LTR system

Abstract

Integration of the HIV cDNA into the host chromosome is a key event in the viral replication cycle. It is mediated by the viral integrase (IN) enzyme, which is an attractive anti-HIV drug target. Here we present the first AFM imaging of IN-mediated DNA integration products in a two-LTR system.

Direct monitoring of the stepwise condensation of kinetoplast DNA networks

“…5A). It has been previously reported that the height of double-stranded DNA measured in AFM images 11 is 0.7 ± 0.2 nm 11,32 , which is lower than the ~ 2 nm diameter of crystallized duplex DNA obtained from X-ray analyses. Accordingly, the heights of the peaks observed here in the uncondensed network may correspond to DNA strands crossing over 2-3 times, as was also suggested by Cavalcanti et al 11 .…”

“…This method enables the monitoring of structural changes at the nano-scale resolution, with no fixation, staining or metal coating of the networks 11,29 . AFM has been previously used in the study of protein-DNA interactions and DNA dynamics [30][31][32] , DNA condensation and compaction [33][34][35][36] , mitochondrial nucleoids 37 , chromatin structure and remodeling [38][39][40][41][42][43][44][45] , as well as in the study of kDNA structure in C. fasciculata 11,20,29,46 , Trypanosoma Cruzi 47 , and other kinetoplastids 48 . Figure 4A-C presents AFM images of uncondensed kDNA networks that were positioned on mica surfaces at different angles, demonstrating the folding of three-dimensional cup-like or basket-like structures into two-dimensional planar kDNA networks on the surface, as has been suggested previously based on electron microscopy.…”