Biochemical characterization of the small isoform of Drosophila melanogaster RECQ5 helicase

Abstract

Recently the gene encoding a member of the RecQ helicase family, RecQ5, was cloned from the fruit fly, Drosophila melanogaster [J.J.Sekelsky, M.H.Brodsky, G.M. Rubin and R.S. Hawley (1999) Nucleic Acids Res., 27, 3762-3769]. The Drosophila RecQ5 transcript is alternatively spliced, like its human counterpart, to yield three protein isoforms. Two of these isoforms are almost identical and have a predicted molecular weight of 54 kDa. The third isoform is larger and contains, in addition to the helicase domain shared by all three isoforms, a long highly charged C-terminal region. A small isoform of the Drosophila RecQ5 protein (RECQ5) has been expressed in Escherichia coli and purified. The purified protein is a single-stranded DNA-stimulated ATPase (dATPase) and a 3'-->5' DNA helicase. Hydrolysis of the nucleotide cofactor is required for unwinding activity and dATP supported the unwinding reaction better than other NTPs. The turnover number for the single-stranded DNA-stimulated dATPase activity was 1380 min(-1), approximately 1.5-fold higher than that observed for the ATPase activity (900 min(-1)). The purified protein catalyzed unwinding of partial duplex substrates up to at least 93 bp, however, unwinding of an 89 bp blunt duplex substrate was not detected.

Figure 1

Analysis of purified RECQ5 protein. (A) Purified RECQ5 was resolved on a 9.6% SDS–polyacrylamide gel and stained with Coomassie blue. Lane 1, 2 µg purified RECQ5; lane 2, 4 µg of purified RECQ5. (B) An identical gel was run using 2 µg of purified RECQ5, transferred to nitrocellulose and probed with antibody directed against RECQ5.

Figure 2

ATP hydrolysis is required for helicase activity. Helicase reaction mixtures (20 µl) contained the 42 bp partial duplex substrate, 1.5 µM RECQ5, 20 mM NaCl, 5 mM MgCl₂ and 3 mM ATP unless otherwise specified. The reactions were initiated by the addition of enzyme and were incubated at 30°C for 30 min. Lanes 1 and 2 were controls and contained no enzyme, the reaction shown in lane 2 was boiled for 3 min; lane 3, complete reaction; lane 4, MgCl₂ was omitted; lane 5, MgCl₂ was omitted and 5 mM EDTA was included; lane 6, ATP was omitted; lane 7, 3 mM ATPγS was included; lane 8, 3 mM ATPγS was substituted for ATP; lane 9, 3 mM AMP-PCP was included; lane 10, AMP-PCP was substituted for ATP; lane 11, 3 mM AMP-PNP was included; lane 12, 3 mM AMP-PNP was substituted for ATP. The substrate and product are shown schematically on the left. An asterisk denotes a radioactive label.

Figure 3

Nucleotide cofactors. Helicase assays were as described in Materials and Methods using the 42 bp partial duplex substrate. Each reaction contained 680 nM RECQ5 and one of the eight nucleotides at a final concentration of 3 mM. The data presented is the average of three independent experiments. Error bars represent the standard deviation about the mean.

Figure 4

The dATPase activity is stimulated by ssDNA. dATPase reactions were as described in Materials and Methods. In each reaction RECQ5 was present at 5 nM, and dATP was included at a final concentration of 2 mM. Circles, M13 ssDNA; squares, poly(dT); inverse triangles, linear dsDNA; triangles, rRNA; diamonds, supercoiled dsDNA. The data represent the average of at least three independent experiments. The equation for a rectangular hyperbola was fitted to the data obtained for M13 ssDNA and poly(dT) (solid lines). Error bars represent the standard deviation about the mean.

Figure 5

RECQ5 unwinds DNA in the 3′→5′ direction. Helicase assays were performed as described in Materials and Methods using the substrate for determining the polarity of unwinding. (A) Schematic depiction of the helicase polarity substrate and the anticipated results. (B) Lanes 1 and 2 contained no helicase, lane 2 was heat denatured by boiling for 3 min; lane 3, helicase I at 14 nM; lane 4, helicase II at 5 nM; lane 5, RECQ5 at 850 nM.

Figure 6

Unwinding of partial duplex substrates. Partial duplex substrates containing three different lengths of the duplex region were used in helicase assays to characterize the unwinding reaction catalyzed by RECQ5. Helicase reaction mixtures were as described in Materials and Methods and unwinding reactions were initiated by the addition of enzyme. Each data point is the average of at least three independent experiments. Circles, unwinding measured in the presence of dATP; squares, unwinding measured in the presence of ATP. Error bars represent the standard deviation about the mean.