Skip to main content
. 2004 Sep;24(17):7324–7330. doi: 10.1128/MCB.24.17.7324-7330.2004

FIG. 3.

FIG. 3.

Srs2 unwinds CTG repeat-containing double-stranded DNA. (A) Schematic diagram of putative Srs2 action on a CTG hairpin in vivo. In the diagram, a newly synthesized CTG tract has folded onto itself to form a hairpin (the template strand has been omitted for clarity). Srs2 (diamond) loads onto the 3′ DNA end and unwinds the structure. The single-stranded product might then reanneal properly to the template strand or might be subject to nuclease digestion, followed by resynthesis of the tract. Either outcome would help eliminate hairpins and thereby reduce the appearance of expansions. (B) Partial DNA duplexes containing CTG repeats can be tested in vitro as potential Srs2 helicase substrates. Srs2 action is envisioned to work as in A. The products of unwinding are single-stranded DNAs which can be readily assayed by gel electrophoresis. (C) Predicted structure of the partially double-stranded substrate. The 3′ tail of the longer strand provides a loading site for the helicase. The duplex contains five CTG repeats to mimic a TNR hairpin. The nine complementary base pairs add thermodynamic stability to the duplex and help ensure that the CTG repeats align as predicted. The arrow shows the predicted junction between single- and double-stranded DNA; 24 nucleotides of the bottom strand should be protected from mung bean nuclease, an enzyme that cleaves single-stranded DNA but not double-stranded regions. (D) Nuclease analysis of DNA structure. The annealed substrate, 5′-end labeled on the longer strand with 32P, was incubated with mung bean nuclease. The products were analyzed on a denaturing 15% polyacrylamide gel. Lanes 1, 2, and 3 contain molecular size markers of 34, 24, and 9 nucleotides, respectively. Lane 4 contains the undigested helicase substrate, and lane 5 contains the product after nuclease digestion. (E) Srs2 helicase activity. The DNA substrate, 5′-end labeled on the shorter strand with 32P, was incubated under standard helicase assay conditions (Materials and Methods) with 50 nM Srs2 for the indicated times. Δ, heat-denatured control.