Use of a restriction enzyme-digested PCR product as substrate for helicase assays

Abstract

DNA helicases play essential roles in many cellular processes. The currently available techniques to generate substrates for helicase assays are fairly complicated and need some expertise not available in all laboratories. Here, a PCR-based method to generate a substrate for a helicase assay is described, and its application for several archaeal, bacterial and viral enzymes is demonstrated.

Figure 1

M.thermautotrophicus MCM can initiate duplex DNA unwinding from a short 3′ single-stranded overhang. Two oligonucleotides were used to generate a helicase substrate with a 4-base ssDNA overhang. Helicase assays were performed as described in Material and Methods in 15 μl reactions with the indicated concentrations of protein and 10 fmol substrate. The ³²P-labeled oligonucleotide is marked with an asterisk. Lane 1, substrate only; lane 2, boiled substrate; lanes 3–5 contain 0.13, 0.40 and 1.2 pmol of proteins (as monomer), respectively. The percent displacement of the labeled oligonucleotide is indicated as %.

Figure 2

Schematic diagram for the procedures developed to generate a PCR-based substrate for helicase assays. Bold arrows are the PCR primers and ³²P is depicted by asterisks.

Figure 3

Substrates for helicase assays generated by PCR. A PCR product and its restriction fragment derivatives are shown. PCR reactions were performed as described in Materials and Methods with ³²P-labeled primer, and products were purified using the QIAquick PCR purification kit (Qiagen) (lane 1) and digest with PstI (lane 2), EcoRI (lane 3) and SmaI (lane 4) restriction enzymes.

Figure 4

PCR-generated substrates can be used for helicase assays. (A) Helicase assays with the M.thermautotrophicus MCM helicase. Helicase assays were performed as described in Material and Methods with 10 fmol of substrate as indicated in the figure. Lanes 1, 6 and 11, substrate only; lanes 2, 7 and 12, boiled substrate; lanes 3–5, 8–10 and 13–15, containing 0.13, 0.40 and 1.2 pmol of proteins (as monomer), respectively. The ³²P-labeled strands are marked with an asterisk. The percent displacement of the labeled oligonucleotide is indicated as %. (B) PCR-generated substrates can be used for helicases with different polarities. Helicases with known 3′→5′ polarity (left panel) or 5′→3′ polarity (right panel). The helicase activity of several bacterial and viral helicases was determined using the PCR-based substrate containing either a 5′ or a 3′ single-stranded overhang. Helicase assays were performed as described in Material and Methods with 10 fmol of each substrate and 1.2 pmol of enzyme. ³²P-labeled strands are marked with an asterisk. Left panel: lane 1, substrate only; lane 2, boiled substrate; lane 3, SV-40 large T-antigen; lane 4, E.coli PriA; lane 5, E.coli Rep helicase; lane 6, E.coli RecG; lane 7, E.coli UvrD. Right panel: lane 1, substrate only; lane 2, boiled substrate; lane 3, E.coli RecQ; lane 4, E.coli RecG. The percent displacement of the labeled oligonucleotide is indicated as %.