FIG 4.

HedH4 excises hedamycin-guanine adducts from DNA and provides cellular resistance to hedamycin toxicity. (A) HED modification of deoxyguanosine in DNA forms a HED-DNA adduct that is hydrolyzed by HedH4 to generate an abasic (AP) site in the DNA and free HED-guanine. The reactions within the dashed line are not catalyzed by HedH4. The AP nucleotide is susceptible to base-catalyzed nicking to form shorter DNA products containing either a 3′-phospho-α,β-unsaturated aldehyde (PUA; β-elimination) or a 3′-phosphate (δ-elimination). The asterisk denotes the original 5′-end of the DNA. (B) Denaturing PAGE of 5′-Cy5-labeled HED-DNA substrate and β- and δ-elimination products after treatment with enzyme or buffer (mock) for 1 h, followed by NaOH to nick the AP site. The HED-DNA reaction only goes to ∼50% completion under our reaction conditions, as shown by the two bands of equal intensity in the mock reaction. (C) HPLC-MS analysis of HED (blue) and the HED-guanine excision product from reaction of HedH4 and HED-DNA (red). Axis represents elution time (x–axis) versus relative abundance from total ion count (y–axis). Insets show mass spectra of each elution peak. (D) Wild-type and mutant HedH4 glycosylase activity for HED-DNA. Spontaneous depurination from a no-enzyme reaction (mock) is shown as a negative control. Data are means ± standard deviations (SD) (n = 3). Curves were fit to a single exponential. Representative data are shown in Fig. S3C. (E) Denaturing PAGE of HED-DNA adducts after 1 h of incubation with either buffer (mock) or bacterial alkylpurine-DNA glycosylases. (F) Denaturing PAGE of 1-h reaction products of E. coli YcaQ and HedH4 with 7mG-DNA (left) and S. bottropensis TxnU4 and HedH4 with TXNA-DNA (right). (G) Structure of NM₈-ICL. (H) Denaturing PAGE of AZB-ICL unhooking by S. sahachiroi AlkZ and HedH4 (left) and NM₈-ICL unhooking by E. coli YcaQ and HedH4 (right). Reactions were treated with buffer (mock) or enzyme for 1 h, followed by alkaline hydrolysis. MA, monoadduct. (I) HED inhibition of E. coli K-12 transformed with hedH4/pSF-OXB1 (constitutively expressed) or empty vector pSF-OXB1. The lag time is defined as the time elapsed before cells start to grow exponentially. Data are means ± SD (n = 3). Growth curves are shown in Fig. S3F and G. Significance values were determined by unpaired t test of the mean lag time values (*, 0.05 ≤ P ≤ 0.01; ***, 0.001 ≤ P ≤ 0.0001). (J) Colony dilution assay for E. coli strains with or without HedH4 exposed to increasing concentrations of HED for 1 h. Surviving fraction (%) is relative to untreated cells. Values are means ± SD (n = 3). Significance values were determined by unpaired t test of the mean sensitivity values (*, 0.05 ≤ P ≤ 0.01; **, 0.01 ≤ P ≤ 0.001).