DNA HELICASE ASSAY: Principle & ACTIVITY of dnaB and assay procedure for polarity with electrophoretic gel pattern.

DNA HELICASE

  The enzyme that harnesses the chemical energy of ATP to separate the two parental DNA strands at the replicating fork is called a helicase. Many DNA helicases have been identified in E. coli cells. The problem is finding which of these is involved in DNA replication. The first three to be investigated the rep helicase, and DNA helicases II and III could be mutated without inhibiting cellular multiplication. This made it unlikely that any of these three enzymes could participate in something as vital to cell survival as DNA replication; we would anticipate that defects in the helicase that participates in DNA replication would be lethal. 

One way to generate mutants with defects in essential genes is to make the mutations conditional, usually temperature sensitive. That way, one can grow the mutant cells at a low temperature at which the mutation is not expressed, then shift the temperature up to observe the mutant phenotype. As early as 1968, François Jacob and his colleagues discovered two classes of temperature-sensitive mutants in E. coli DNA replication. Type 1 mutants showed an immediate shut-off of DNA synthesis on raising the temperature from 308C to 408C, whereas type 2 mutants showed only a  gradual decrease in the rate of DNA synthesis at elevated temperature. 

One of the type 1 mutants was the dnaB mutant; DNA synthesis in E. coli cells carrying temperature-sensitive mutations in the dnaB gene stopped short as soon as the temperature rose to the non permissive level. This is what we would expect if dnaB encodes the DNA helicase required for replication. Without a functional helicase, the fork cannot move, and DNA synthesis must halt immediately. Furthermore, the dnaB product (DnaB) was known to be an ATPase, which we also expect of a DNA helicase, and the DnaB protein was found associated with the primase, which makes primers for DNA replication. 

All of these findings suggested that DnaB is the DNA helicase that unwinds the DNA double helix during E. coli DNA replication. All that remained was to show that DnaB has DNA helicase activity. Jonathan LeBowitz and Roger McMacken did this in 1986. They used the helicase substrate shown in Figure, which is a circular M13 phage DNA, annealed to a shorter piece of linear DNA, which was labeled at its 59-end. Figure also shows how the helicase assay worked. LeBowitz and McMacken incubated the labeled substrate with DnaB, or other proteins, and then electrophoresed the products. If the protein had helicase activity, it would unwind the double-helical DNA and separate the two strands. Then the short, labeled DNA would migrate independently of the larger, unlabeled DNA, and would have a much higher electrophoretic mobility. Figure shows the results of the assay. DnaB alone had helicase activity, and this was stimulated by DnaG, and by SSB, a single-stranded DNA-binding protein that we will introduce next. Neither DnaG nor SSB, by themselves or together, had any DNA helicase activity. Thus, DnaB is the helicase that unwinds the DNA at the replicating fork.

Principle of assay. LeBowitz and McMacken made a helicase substrate (top) by 32P-labeling a single-stranded 1.06-kb DNA fragment (red) at its 59-end and annealing the fragment to an unlabeled single-stranded recombinant M13 DNA bearing a complementary 1.06-kb region. The dnaB protein, or any DNA helicase, can unwind the double-stranded region of the substrate and liberate the labeled short piece of DNA (red) from its longer, circular partner. Bottom: Electrophoresis of the substrate (lane 1) yields two bands, which probably correspond to linear and circular versions of the long DNA annealed to the labeled, short DNA. Electrophoresis of the short DNA by itself (lane 2) shows that it has a much higher mobility than the substrate (see band labeled “Product”).

Helicase assay results. LeBowitz and McMacken performed the assay outlined in above assay with the additions (DnaB, DnaG, and SSB) indicated at top. The electrophoresis results are given at bottom. Lane 1 is a control with the unannealed, labeled short DNA to show its electrophoretic behavior (arrow). Lane 3 shows that DnaB has helicase activity on its own, but lanes 4 and 5 demonstrate that the other proteins stimulate this activity. On the other hand, lanes 7–9 show that the other two proteins have no helicase activity without DnaB

NOTE: The polarity of helicase activity has also been determined.

To determine the polarity of DNA helicase action, a substrate is employed that contains two duplex regions of distinct length flanking a central ssDNA region. Both short DNA strands (designated X oligo and Y oligo) are radioactively labeled. DNA helicase unwinding in the 5 ' —*3' direction would release the X oligonucleotide, and unwinding in the 3 ' —»5' direction would release the Y oligonucleotide. 

For helicases involved in DNA replication, the polarity of the reaction is strongly indicative of helicase placement on the leading (a 3 ' -5 'polarity) or lagging (a 5 ' +3 'polarity) strand.The polarity assay. To determine the polarity of DNA helicase action, a substrate is employed that contains two duplex regions of distinct length flanking a central ssDNA region. Both short DNA strands (designated X oligo and Y oligo) are radioactively labeled. DNA helicase unwinding in the 5 ' —*3' direction would release the X oligonucleotide, and unwinding in the 3 ' —»5' direction would release the Y oligonucleotide.

A brief procedure explaining the assay for polarity of Helicase