1. The SE domains from BLM/Sgs1 orthologs show DNA SE activity.
The SE domains from BLM/Sgs1 orthologs show DNA SE activity. (A) The SE assay is illustrated at the top of the panel. Reactions contained the indicated SE domain proteins at 0 (−), 50, 200 or 400 nM plus 2 nM forked DNA (where oligo #1 is 32P‐labelled) plus 10 nM oligo #1. Substrate DNAs were incubated with GST at 50, 200 or 800 nM as negative control. The reactions were incubated at 37°C for 30 min under standard conditions and the products were analysed by 8% PAGE and phosphorimaging. The first lane (Δ) contains 32P‐labelled oligo #1 as marker. (B) Sgs1103−322 (0, 100, 200, 400, 800 and 1600 nM) was used in an SE assay using blunt‐ended substrate as indicated in the reaction at the top of the panel. Reactions contained 1 nM duplex DNA plus the indicated amounts of oligo 3. Assays were performed as in (A) except for use of 10% PAGE. (C) Sgs1103−322 was assayed using 5′‐tail duplex DNA as substrate as in (B). (D) Sgs1103−322 was assayed using 3′‐tail duplex DNA as substrate as in (B). (E) The SE reactions shown in (B–D) (20 nM unlabelled oligo) were quantified and are presented as a function of protein concentration. Sequences of the indicated oligos are presented in Supplementary Table 1. Throughout, all proteins are His6‐tagged. Asterisks represent positions of 32P‐labelling.
Chi‐Fu Chen, and Steven J Brill EMBO J. 2010;29:
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