Using 3′- or 5′-end-labelled DNA substrates to identify the termini of unwound DNA (anti-biotin antibody-conjugated gold beads bound to the DNA ends are visible as electron-dense particles in electron micrographs), Taylor and Smith determined the strand polarity of the unwinding structures produced by a RecB-helicase-defective mutant, RecBK29QCD. The electron micrographs showed a ssDNA loop and only one, 5′-ended, ssDNA tail with equivalent lengths. So, the authors concluded that the inactive RecB subunit remained at the 3′ end where it initially bound, producing a loop but no second tail. The rate of unwinding was nearly identical to that of wild-type RecBCD, indicating that RecD helicase, which binds to the 5′ end, is a faster helicase than RecB and of opposite polarity.
To test the contribution of the RecB helicase, Taylor and Smith used RecBC enzyme (lacking RecD) and found that it produced Y-shaped molecules with two equal-length ssDNA tails but without loop formation, as expected for a single-helicase enzyme. In addition, a RecD-defective mutant, RecBCDK177Q, produced 'loop two-tail' unwinding structures, but with a 3′-ended long ssDNA tail and a very short 5' tail, implying a strand polarity opposite to that of the wild-type enzyme. The inactive RecD subunit remained bound at or near the 5′-ended strand where it formed a loop and a nearly equal-length ssDNA tail on the opposite strand. Either removal of the RecD subunit or inactivation of its helicase activity, by using the RecBCDK177Q mutant, resulted in an unwinding rate ∼20% of that of the wild-type enzyme — confirming that RecB is the slower helicase that functions on the 3′-ended strand.
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