Figure 3.

YcaQ unhooks nitrogen mustard ICLs. (A) 5-atom nitrogen mustard (NM₅) ICL formed between mechlorethamine and guanines located on opposite DNA strands. (B-C) Denaturing PAGE of NM₅-ICL substrate, monoadduct, and nicked AP-DNA products after treatment with WT or mutant YcaQ (B), or with YcaQ, human AAGΔ83, E. coli AlkA, or S. pombe Mag1 (C), followed by treatment with EndoIV. The percent of unhooked ICL is quantified below the gel. Images are false colored overlays of individual FAM and Cy5 scans of the gel, which are shown in Supplementary Figure S3. (D) Denaturing PAGE showing time-dependence of AlkZ and YcaQ unhooking of NM₅-ICLs with a hydroxide workup. The plot quantifies the decrease in the fraction of NM₅-ICL (mean ± SEM, n = 3). (E) Quantification of the fraction of monoadduct (MA) and β,δ-elimination (nicked AP-DNA product) from the mock (left), AlkZ (right), and YcaQ (middle) reactions from the gel in panel D.