Figure 1. Dna2 initiates the resection of clean DSB ends via cleavage of 5′ strand DNA. (A) A one-end 5′ 32P-labeled 2 kb DNA fragment (red star, 32P; purple circle, biotin) was incubated in NPE at room temperature. Reactions were terminated at the indicated times and resection products were resolved on a 16% polyacrylamide-urea gel. The top band represents the original DNA substrate that was ‘trapped’ in the loading wells of the gel. (B) Effects of Dna2 depletion on the resection of the radiolabeled DNA substrate depicted in (A) in the extract. (C) Purified recombinant Flag-Dna2(WT), Flag-Dna2(D278A) and Flag-Dna2(K655E) expressed in insect cells. (D) Comparison of the flap endonuclease activity of Flag-Dna2(WT), Flag-Dna2(K655E) and Flag-Dna2(D278A) towards a dsDNA substrate with a 5′ ssDNA flap in vitro. (E) Rescue of short 5′ endocleavage products in the Dna2-depleted extract by recombinant Flag-Dna2(WT) protein. (F) Comparison of the ability of recombinant Flag-Dna2(WT), Flag-Dna2(K655E) and Flag-Dna2(D278A) in restoring resection initiation in the Dna2-depleted extract.
Image published in: Paudyal SC et al. (2017)
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