XB-IMG-170839
Xenbase Image ID: 170839
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Figure S1. Schematic of ICL plasmids and preparation of SJG-136 treated plasmids, Related to Figure 1
(A) SJG-136 preferentially reacts with DNA at guanine residues at 5’ purine–GATC–pyrimidine sequences to form
ICLs.
(B) Sequences of trimethylene and SJG-136 ICL oligos that were ligated into pBS to construct plasmids with a
single site-specific ICL (left). Schematic of the resulting ICL plasmid. The binding site for Bio-LacR used for
plasmid pull-down experiments, and primers used for qPCR to calculate ICL repair are illustrated (right).
(C) Schematic representation of pBS treated with SJG-136. The 7 sequences corresponding to SJG-136 ICL
forming sites are illustrated. The single SJG-136 reaction site that overlaps with a BamHI recognition sequence
2
is specified. The binding site for Bio-LacR and primers used for quantification of plasmids in plasmid pulldown
experiments are indicated.
(D) Accumulation of ICLs on SJG-136 treated plasmids assessed using BamHI digestion. Plasmids were run on
ethidium-bromide agarose gel before (upper panel) or after BamHI digestion (lower panel). With increasing
doses of the drug, plasmids become refractory to BamHI digestion.
(E) The number of ICLs induced on plasmids treated with SJG-136 was estimated from quantification of BamHI
digestion products. Results represent quantification from 3 independent experiments ± SD.
(F) Representative figure of the quantification of plasmids recovered from extracts after pull-down experiments
using qPCR. Results represent technical triplicate ± SD from a single representative experiment. This analysis
was used as a loading control to ensure equivalent amounts of plasmids were loaded on gels for Western blot.
(G) SJG-136 treated plasmids were incubated in HSS, and soluble extracts were analyzed by Western blot. Image published in: Kato N et al. (2017) Copyright © 2017. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View |