Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2016 Jul 27;44(16):7866–7883. doi: 10.1093/nar/gkw661

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2016. 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 License (http://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

PMC Copyright notice

Figure 6. — Effect of RecA and Ssb on IS608 excision and integration of in vitro. (A) RecA binding and competition with TnpA_IS₆₀₈: Binding of increasing concentrations of RecA (1, 2.5, 5, 10 μM) to labelled ssLE and unlabelled ssRE in the presence of ATP (i, lanes 2–5) or ATPγS (ii, lanes 2–5). TnpAIS608 (0.1 and 1 μM) was added after 15 min of preincubation with RecA (i and ii, lanes 6–7). Synaptic complex TnpA_IS₆₀₈–LE–RE with 1 μM TnpA_IS₆₀₈ (i and ii, lane 8) is formed in the presence of ATP and ATPγS respectively. Binding of increasing concentrations of RecA (1, 2.5, 5, 10 μM) to labelled ss target and unlabelled ssRE-LE junction in the presence of ATP (iii, lanes 2–5) or ATPγS (iv, lanes 2–5). TnpA_IS₆₀₈ (0.1 and 1 μM) was added after 15mins of preincubation with RecA (iii and iv, lanes 6–7). Integration product with 1 μM TnpA_IS₆₀₈ (iii and iv, lane 8) is formed in the presence of ATP and ATPγS respectively. (B) Effect of RecA on excision and integration: excision and integration after 15 min of preincubation with increasing concentrations of RecA in the presence of ATP (i, lanes 2–6 and iii, lanes 2–6) or ATPγS (ii, lanes 2–6 and iv, lanes 2–6) respectively. (C) Ssb binding and competition with TnpA_IS₆₀₈: Binding of increasing concentrations of Ssb (0.5, 1.25, 2.5 and 5 μM) to labelled ssLE80 (lanes 2–5) or labelled ssLE100 (lanes 11–14) and unlabelled ssRE respectively. TnpA_IS₆₀₈ (0.1 and 1 μM) was added after 15 min of preincubation with Ssb (lanes 6–7). Synaptic complexes TnpA_IS₆₀₈–LE–RE with 1 μM TnpA_IS₆₀₈ are shown (lanes 8 and 16) respectively. Red arrow (lane 15) shows the position of co-complex TnpA_IS₆₀₈–LE–RE-Ssb. Binding of increasing concentrations of Ssb (0.5, 1.25, 2.5 and 5 μM) to labelled ss target and unlabelled ssRE-LE junction (lanes 18–21). TnpA_IS₆₀₈ (0.1 and 1 μM) was added after 15 min of preincubation with Ssb (lanes 22–23). Lane 24 includes 1 μM TnpA_IS₆₀₈ and labelled target and unlabelled junction. (D) Effect of Ssb on excision and integration: excision with labelled LE80 or LE100 and unlabelled RE56 after 15 min of preincubation with increasing concentrations of Ssb (lanes 2–6) and (lanes 8–12) respectively. Integration of cold RE–LE junction into labelled ss target in the absence (lane 14) and after 15 min of preincubation with increasing concentrations of Ssb (lanes 15–18). Integration of labelled RE–LE junction into cold ss target in the absence (lane 20) or in the presence of 5 μM Ssb (lane 21).