PcrA^s_FH preferentially unwinds DNA substrates with a 3′ ss terminus.

<p>(A) DNA helicase activity is not significantly influenced by the position of the fluorescent label in the DNA substrate. DNA helicase assays were performed with PcrA^s_FH and either substrate ‘a’ (6-FAM-labelled at either the 3′ end [lanes 1 and 2] or 5′ end [lanes 3 and 4] of strand 1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070870#pone-0070870-g001" target="_blank">Fig. 1</a>]) or substrate ‘b’ (6-FAM-labelled at either the 3′ end [lanes 5 and 6] or 5′ end [lanes 7 and 8] of strand 1). The DNA helicase reactions were carried out in volumes of 10 µl and contained DNA substrate (at 8 nM) and either 0 nM (marked ‘−’, lanes 1, 3, 5 and 7) or 12 nM of protein (‘+’, lanes 2, 4, 6 and 8). After the reaction (10 min at 37°C), the samples were deproteinized, electrophoresed through a native 12% polyacrylamide gel, and analyzed by fluorometry. The positions of the substrates (s) and reaction products (p) are indicated at the right-hand side of the gels. (B) PcrA_s^FH preferentially unwinds branched DNA substrates that carry a 3′ ss terminus. The DNA helicase activity of PcrA^s_FH was tested on branched DNA substrates carrying either a 3′ ss terminus (substrate ‘d’, lanes 1 and 2), a 5′ ss terminus (substrate ‘e’, lanes 3 and 4) or blunt ends (substrate ‘f’, lanes 5 and 6). The sequences and structures of the different substrates are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070870#pone-0070870-g001" target="_blank">Fig. 1</a>. The reactions were performed in a similar fashion as described in (A).