Our results suggest a new model where two IN dimers individually assemble on U3 and U5 ends prior to the non-covalent juxtaposition of two viral DNA ends, producing the synaptic complex. = (for rhodamine 110 was 0.91 in 10 mM HEPES, pH 7.5 made up of 15% ethanol48 and fluorescein was 0.95 in 0.1 N NaOH49. In-gel FRET data analysis of SC and STC To determine FRET signal intensities, three sets of reaction mixtures were prepared: 1) contains 3 nM of Cy3-U5 (donor fluorophore) substrate; 2) contains 1.5 nM of each Cy3-U5 and Cy5-U5 (donor-acceptor fluorophore pair); 3) contains 3 nM Cy5-U5 (acceptor fluorophore)(Fig. from the 3 OH ends and performs the concerted insertion of two viral DNA ends into target DNA. IN remains associated with the concerted integration product, termed the strand transfer complex. The synaptic complex and strand transfer complex can be isolated on native agarose gels for biochemical and biophysical analyses. In this report, in-gel fluorescence resonance energy transfer measurements exhibited Kinetin that this energy transfer efficiencies between the juxtaposed Cy3 and Cy5 5-end labeled viral DNA ends in the synaptic complex (0.68 0.09) was significantly different than observed in the strand transfer complex (0.07 0.02). The calculated distances were 46 3 ? and 83 5 ?, respectively. DNaseI footprint analysis of the complexes revealed that IN protects U5 and U3 DNA sequences up to ~32 bp from the end, suggesting two IN dimers were bound per terminus. Enhanced DNaseI cleavages were observed at nucleotide positions 6 and 9 from the terminus on U3 but not on U5 suggesting independent assembly events. Protein-protein cross-linking of IN within these complexes revealed the presence of dimers, tetramers, and a larger-size multimer ( 120 Kd). Our results suggest a new model where two IN dimers individually assemble on U3 and U5 ends prior to the non-covalent juxtaposition of two viral DNA ends, producing the synaptic complex. = (for rhodamine 110 was 0.91 in 10 mM HEPES, pH 7.5 made up of 15% ethanol48 and fluorescein was 0.95 in 0.1 N NaOH49. In-gel FRET data analysis of SC and STC To determine FRET signal intensities, three sets of reaction mixtures were prepared: 1) contains 3 nM of Cy3-U5 (donor fluorophore) substrate; 2) contains 1.5 nM of each Cy3-U5 and Cy5-U5 (donor-acceptor fluorophore pair); 3) contains 3 nM Cy5-U5 (acceptor fluorophore)(Fig. 3). As an activity control, 3 nM of unlabeled U5 was also used concurrently. For FRET signal measurements, the gel was scanned in a Typhoon Trio variable image laser scanner with green laser (532 nm) and a 580 nm emission filter with band pass 30 nm for detection of Cy3 fluorophore (donor) quenching. For the sensitized FRET signals, the gels were scanned with a green laser (532 nm) and a 670 nm emission filter with band Parp8 pass 30 nm. The latter scan produced the sensitized emission of Cy5-U5 (acceptor) via resonance energy transfer. The fluorescence intensity of SC and STC were determined by using ImageQuant 5.2 software. The quenched FRET signals were determined from the extent of donor fluorescence quenching in the complex made up of the acceptor compared with donor fluorescence in absence of acceptor fluorophore. With proper control experiments, the sensitized FRET signal for acceptor fluorophore was also calculated. The gel was scanned for acceptor emission at 670 nm (ID, IAD and IA for sensitized FRET)(Fig. 3a) and donor emission (ID and IDA for quenched FRET)(Fig. 3b) by excitation of the donor. ID is the contribution of fluorescence emission intensity of the donor fluorophore at 670 nm due to excitation at 532 nm in absence of acceptor fluorophore. IA and IAD are the fluorescence emission intensities of acceptor fluorophore at 670 nm in absence and presence of donor fluorophore, respectively, when excited at 532 nm. ID and IDA are the fluorescence emission intensities at 580 nm of donor fluorophore in absence and presence of acceptor fluorophore, respectively, when excited at 532 nm. After the laser scans, the same gel was stained with SYBR Gold Stain (Fig. 3c) for quantitative analysis of the FRET data. Kinetin Fluorescence intensities were normalized for the differences in amounts of donor-only ([complex]D) and donor-and acceptor-labeled complex ([complex]DA) by multiplying with [complex]D/[complex]DA. In Fig. 3d, quenched FRET was decided for SC, produced after 20 min at 37C, as the difference between corrected fluorescence intensities of donor fluorophore in absence and presence of acceptor fluorophore. Similarly, sensitized FRET is usually equal Kinetin to IAD? IA ? ID (corrected intensities). When calculating FRET intensities and hence energy transfer efficiency (E), we account for the Kinetin appropriate concentrations of donor and acceptor fluorophore-labeled DNA substrates present in the reaction mixture. Formation of SC with Cy3-U5 and Cy5-U5 produces four kinds of complexes. They are: Cy3-U5 & Cy3-U5, Cy3-U5 & Cy5-U5, Cy5-U5 & Cy3-U5, and Cy5-U5 & Cy5-U5, all with equal probability of being produced. Only two complexes (donor-acceptor and acceptor-donor) are responsible for energy transfer. With impartial experiments, Fig. 3e shows the quantitative in-gel FRET analysis for STC produced after 120 min at 37C. The same IN concentrations for SC (Fig. 3a to 3d) were used for the in-gel FRET analyses of STC. The F?rster distance R0 was calculated for donor-acceptor pairs by the method of Wu and Brand 50. Quantum yields of Cy3-U5 were nearly identical in the electrophoresis Tris-Borate-EDTA (TBE) buffer and in 0.7% agarose in Kinetin TBE buffer.