Fig. 5: The MD integrates and regulates Upx, with activation by a phage protein and targeting of structured nucleic acids. | Nature Communications

Fig. 5: The MD integrates and regulates Upx, with activation by a phage protein and targeting of structured nucleic acids.

From: Structural and mechanistic insights into the dual-nuclease defense protein Upx as an anti-phage system

Fig. 5: The MD integrates and regulates Upx, with activation by a phage protein and targeting of structured nucleic acids.The alternative text for this image may have been generated using AI.

a Domain organization of Upx showing NTD, MD, and CTD. b–c Interaction assays between MD and NTD (b) or MD and CTD (c). d Electrophoretic mobility shift assays (EMSA) of 5’ Cy3-labeled ssDNA (60 nt) incubated with full-length Upx or its truncated variants. All assays were performed in triplicate. e Pulldown assay showing interaction between Upx and gp16.Each assay was performed in triplicate. f gp16 relieves MD-mediated inhibition of NTD nuclease activity, assayed using 5’ Cy3-labeled ssDNA (60 nt). All cleavage products were analyzed by 13% denaturing urea-PAGE and visualized by fluorescence imaging. Each assay was performed in triplicate. g–k Cleavage activity of Upx on different structured DNA substrates, including 5’ overhang DNA, 3’ overhang DNA, partial duplex DNA, and forked DNA. Asterisks denote the Cy5-labeled strand. All cleavage products were analyzed by 13% denaturing urea-PAGE and visualized by fluorescence imaging. Each assay was performed in triplicate. l Cleavage assays were performed using the unpaired 3’ single-stranded regions within both D-loop and R-loop structures. Asterisks denote the fluorescently labeled strands (Cy3, green; Cy5, magenta). Each assay was performed in triplicate. m Cleavage assays were performed using D-loop structures labeled with 5’ Cy3 at the terminus. The cleavage products were analyzed by 6% native urea–PAGE and visualized by fluorescence imaging. Each assay was performed in triplicate.

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