Rapid purification of helicase proteins and in vitro analysis of helicase activity

Abstract

Most processes involving an organism's genetic material, including replication, repair and recombination, require access to single-stranded DNA as a template or reaction intermediate. To disrupt the hydrogen bonds between the two strands in double-stranded DNA, organisms utilize proteins called DNA helicases. DNA helicases use duplex DNA as a substrate to create single-stranded DNA in a reaction that requires ATP hydrolysis. Due to their critical role in cellular function, understanding the reaction catalyzed by helicases is essential to understanding DNA metabolism. Helicases are also important in many disease processes due to their role in DNA maintenance and replication. Here we discuss ways to rapidly purify helicases in sufficient quantity for biochemical analysis. We also briefly discuss potential substrates to use with helicases to establish some of their critical biochemical parameters. Through the use of methods that simplify the study of helicases, our understanding of these essential proteins can be accelerated.

Figure 1. The pTYB4-His-TraI plasmid

Schematic of the pTYB4-His-TraI plasmid with several unique restriction sites noted. The traI gene is shown in dark blue, the intein-CBD sequence is depicted in cyan, and the 8-His tag is depicted in red. In addition, the origin of replication (Ori), the ampicillin resistance gene (AmpR) and the lacI gene are shown.

Figure 2. A schematic view of purification using pTYB4-His and results from TraI purification

a. Overview of the purification procedure: The fusion protein is bound to a cobalt affinity column (TALON^®) depicted in pink. The protein, including both tags, is eluted from the cobalt affinity column using an imidazole wash and directly bound to a chitin column depicted in light grey. After addition of the reducing agent DTT to promote intein-directed cleavage of the target protein from the affinity tags and incubation at 4°C, the final product is eluted from the chitin column leaving the intein-CBD-His tag bound to the beads. b. Coomassie blue stained SDS polyacrylamide gel from a TraI rapid purification. In each lane 5 μg of total protein has been loaded. Lane 1, crude lysate; lane 2, cleared lysate; lane 3, TALON^® flow through; lane 4, TALON^® column elution; lane 5, chitin column flow through; and lane 6, chitin elution containing purified TraI.

Figure 2. A schematic view of purification using pTYB4-His and results from TraI purification

a. Overview of the purification procedure: The fusion protein is bound to a cobalt affinity column (TALON^®) depicted in pink. The protein, including both tags, is eluted from the cobalt affinity column using an imidazole wash and directly bound to a chitin column depicted in light grey. After addition of the reducing agent DTT to promote intein-directed cleavage of the target protein from the affinity tags and incubation at 4°C, the final product is eluted from the chitin column leaving the intein-CBD-His tag bound to the beads. b. Coomassie blue stained SDS polyacrylamide gel from a TraI rapid purification. In each lane 5 μg of total protein has been loaded. Lane 1, crude lysate; lane 2, cleared lysate; lane 3, TALON^® flow through; lane 4, TALON^® column elution; lane 5, chitin column flow through; and lane 6, chitin elution containing purified TraI.

Figure 3. Two circular substrates for helicase assays

a. A 91-mer oligonucleotide is annealed to M13 ssDNA as described in the text. The 3′-end is extended by two bases using [³²P]dCTP and the Klenow fragment of DNA polymerase I to yield the 93 base pair partial duplex substrate. b. A plasmid containing several Nt.BbvCI sites is nicked to completion with Nt.BbvCI. The small oligonucleotide fragments are heat denatured and removed leaving a gapped plasmid. The gap-containing strand is labeled using [³²P]dCTP, dTTP and the Klenow fragment of DNA polymerase I.

Figure 4. Substrate for determining the directionality of a helicase

A 91-mer oligonucleotide is annealed to M13 ssDNA as described in the text. This partial duplex DNA is digested with BspDI to generate a linear DNA molecule with double stranded regions of different length at either end as shown. The overhangs at the end of the DNA are not drawn to actual size. The resulting overhang ends and the 3′-end of the original oligonucleotide are filled in or extended using [³²P]dCTP, dGTP and the Klenow fragment of DNA polymerase I to produce the labeled DNA molecule shown.