DNA Helicase: Function/role




I.                   Role

The role of helicases is to unwind the duplex DNA in order to provide a single-stranded DNA for replication, transcription, and recombination for instance. [1]


Figure1: Helicase in DNA replication [2].





††† II.††††††††† Function


Brief overview: DNA helicase is a molecular motor-protein that separates or unzips two strands of DNA. It runs along the DNA to unzip it, and while doing so, it converts chemical energy into mechanical energy. It couples conformational changes induced by ATP or NTP binding and hydrolysis of the duplex nucleic acid. [3]


Link to a helicase animation showing the unzipping of duplex DNA:


†††††††††† http://www.scianafilms.com/html/animation/features/t7/index.htm



Note: Scientists think that some DNA helicases function as hexameric rings. Despite many studies scientists are unsure about its operating mechanism, partly due to the lack of good experimental technique.[3]


One group of scientists studied the unzipping mechanism using Fluorescent Resonance Energy Transfer (FRET). [5]


Strategy behind FRET

In a single molecule FRET, two fluorescent dyes are attached to a biological molecule. The interaction between the two fluorescent dyes can tell how far apart they are, and this distance can in turn be used to measure the shape changes of the a single molecule during its function.


FRET in the DNA Unzipping Experiment

So in the DNA unzipping experiment, two dies are attached to the part of DNA where the unzipping begins. Then as the unzipping is initiated by the helicase, the distance between the two dyes increases and the FRET decreases. Importantly, the pauses and initiation of unzipping were detected for the first time for any helicase using this technique. Also, the mechanisms were deduced with the observations using FRET.


Figure 2: close look at DNA-helicase complex






III.†††††††††††† Helicase Mechanism (T7 Helicase)


Part I Structure and Mechanism

One surprising characteristic of the structure is the asymmetry of the hexameric ring. [6]


Figure3: This figure shows the polypeptide backbone of the helicase. Top of side and side view of hexameric structure. Asymmetry can be seen by examining the position of the attached gold atom.



This asymmetry in figure has major implications for the mechanism of the enzyme. It means that nucleotide-binding sites, which are at the interface between the subunits) are different, and will be likey to have differing affinities for the NTPs. Of the six nucleotide-binding sites, electron density which corresponds to bound nucleotide was only seen in four positions. These four positions are two sets of two equivalent sites. Of the four nucleotide-containing sites two have a strong electron density, suggesting an NTP-binding site. But the other two binding sites have weaker electron density, consistent with NDP+Pi binding sites.



Figure4: this shows the proposed binding-mechanism for the T7 helicase (right) compared to the F1-ATPase. Shown black are the non-catalytic alpha subunits of F1-ATPase.



In figure 4, the states of the tree sites may interconvert from NTP-bound to empty as nucleotide hydrolysis occurs. This would allow NTP-hydrolysis to run around the hexameric ring as shown below.



Animation 1: shows the proposed-binding change mechanism for the T7 helicase (right) compared to the F1-ATPase mechanism (left).


Below is a representation of the state of hexameric ring that is generated by NTP-hydrolysis, which runs around.


Animation 2: shows the state of the enzyme as NTP-hydrolysis takes place





Part II Inchworm mechanism


Most helicase require a ssDNA (tail) that is adjacent to a dsDNA region in order to initiate unwinding of the duplex. Hence, the ssDNA serve as a loading strand and a starting point to unwind duplex DNA. But, some helicases can operate at blunt end.


Inchworm mechanism:

This mechanism proposes that there are two binding sites for DNA on the helicase. One binding remains in contact with the DNA, while the other moves a few inches forward along the lattice to bind a new section of the DNA; This happens as a result of ATP binding and hydrolysis.



Figure 5: shows a 90 degree rotation of the PcrA helicase to duplex DNA binding




Inchworm Model for PcrA Helicase Activity:

As opposed to DnaB helicase, PcrA is monomeric enzyme that is proposed to contain two binding site for ssDNA and dsDNA. Its catalyzed activity is proposed to consist of two distinct but coupled activities:

  • Translocation of a single base of DNA
  • Active distortion of the duplex



Figure 6: Inchworm model for PcrA helicase activity



Additional picture showing the substrate bound to PcrA helicase below.



Figure 7: shows substrate bound to PcrA helicase



Now: see a movie (PcrA helicase substrate complex)


††††††††††† Wigley Lab Home Page


See another movie (The Mexican Wave) showing the translocation of a ssDNA inside the PcrA helicase:


††††††††††† Wigley Lab Home Page


Part III: what exactly occurs in term of forces?

Helicases use the energy of ATP to break (melt) the hydrogen bonds and dissipate the forces (hydrophobic effect, electrostatic interaction) that hold the strands together in the duplex DNA. [1]





Figure 8: Unwinding of DNA [7]


Mechanism of T7 Primase/Helicase:


Researchers think that the T7 helicase is dependent on primase, meaning that the primase/helicase complex from the T7 bacteriophage is required for unwinding duplex DNA [4]. This next movie is based on X-ray crystallized structure of the primase and helicase domains, and it shows how both work together. It consists of two parts:

  • The possible mechanism of DNA unwinding by the helicase domain
  • The mechanism of RNA primer synthesis by the primase binding domain.

But for the purpose if this presentation we will ignore the primer synthesis part of the movie.






1. Kornberg, Arthur, and Tania A. Baker. DNA Replication. New York: W. H. Freeman and Company, 1992.

2. <http://www.sp.uconn.edu>.

3. Tackett, Alan J., Patrick D. Morris, Regina Dennis, Thomas E. Goodwin, and Kevin D. Raney. (2001), Biochemistry, pp. 543-548.

4. Sciana Film Works. <http://www.scianafilms.com/html/animation/features/t7/index.htm>.

5. The department of Physics at the University of Illinois at Urbana-Champaign. 11 Nov. 2002. <http://www.physics.uiuc.edu>.

6. Cancer Research UK, Molecular Enzymology, and London Research Institute. <http://sci.cancerresearchuk.org>.

7. Doc Kraizerís Microbiology Home Page. 30 Oct. 2001. <http://www.cat.cc.md.us>.



Biochemistry Project

By Wilfried Foadey

5 November 2004