Skip to main content
. 2021 Nov 26;12:6942. doi: 10.1038/s41467-021-27284-7

Fig. 4. Measurement of adduct excision activity.

Fig. 4

a Sequence specificity of SCPCHD natural products. Modified nucleotides are indicated with arrows. The central 2′-deoxyadenosine nucleotide is modified by DSA with relatively low frequency. b Separation of full-length DNA substrate (25-mer) from alkali-cleaved product (12- or 13-mer) by denaturing gel electrophoresis. Oligonucleotide substrates were incubated with or without (mock) enzyme for 30 s before reactions were quenched with sodium hydroxide and heat to denature both the enzyme and the DNA and to cleave the DNA at the AP site. The high melting temperature of the GC-rich substrate prevented complete denaturation of the DNA duplex, producing a smeared substrate band. The unique sequence specificities of YTM, CC-1065, and DSA for different 2′-deoxyadenosine nucleotides result in generation of alkali-cleaved products of different lengths. The lower specificity of DSA allows for modification of one of two nucleotides, each corresponding to a different product band. Experiments were performed in triplicate. c Single-turnover excision of SCPCHD adducts. Reactions contained 1 μM enzyme and 100 nM DNA. With all substrates, both AlkD and YtkR2 approached the endpoints of the reactions by the first time point, potentially reducing or masking differences in excision rates between the adducts and between the enzymes. d Multiple-turnover excision of SCPCHD adducts. Reactions contained 10 nM enzyme and 100 nM DNA. e Incision of AP-DNA. SCPCHD adducts were pre-incubated with AlkD, YtkR2, or C10R5 to generate AP-DNA for subsequent incision by EndoIV. THF-DNA and 3mAde nucleobase were also pre-incubated with each homolog or without enzyme (mock). Incomplete excision of YTM-Ade by C10R5 during the pre-incubation reaction reduced the amount of AP-DNA (Supplementary Fig. 7), limiting the possible fraction product. The affected quantity is indicated with an asterisk. Of the AP-DNA produced, 98% was subsequently incised by EndoIV. Data in c–e are presented as the mean ± SD from three replicate experiments. Source data are provided as a Source Data file.