Unwinding the Double Helix: Meet DNA Helicase - Evolution News & Views

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Unwinding the Double Helix: Meet DNA Helicase

In previous articles (see here, here and here), I've been reviewing the molecular nano-machinery needed for the replication of DNA. Before DNA polymerase is able to synthesize the new complementary strands, it needs to be given access to the nucleotides of the single-stranded template DNA. The internal base pairing in the double helix must therefore be broken and the helix unwound. Generally, the initial opening of the double helix (at the origin of replication) is performed by an initiator protein (Stenlund, 2003). DNA helicases can melt base pairs using the energy released during the process of binding, hydrolysis and release of ATP.

DNA helicase travels ahead of the replication fork, continuously opening and unwinding the DNA double helix to provide the template needed by the DNA Polymerase. With a rotational speed of up to 10,000 rotations per minute, the helicase rivals the rotational speed of jet engine turbines. When I first encountered and studied the mechanisms of DNA replication in my early undergraduate days, I was stunned by its complexity and elegance. I later came to the realization, however, that my initial conception of the sophistication of these molecular machines was a gross underestimation. The closer I inspected the nanomachinery responsible for information processing in the cell, the more I felt a sense of astonishment and marvel. You could write an entire book about each and every one of the numerous nanomachines needed for successful DNA replication. Indeed, such a book on DNA helicases and related DNA motors was recently published.

The Structure and Function of DNA Helicase

DNA helicases are generally highly sophisticated ring-shaped multimeric ATP-fuelled nanomachines, with molecular weights of more than 300kDa. They are members of the AAA+ protein superfamily, being characterized by a catalytic and nucleotide-binding site known as the AAA+ domain (Snider et al., 2008).

DNA helicases are composed of six subunits that make up a hexameric ring structure, as shown in the animation above. In papillomavirus, the subunits are made up of just one protein, E1 (Hughes and Romanos, 1993). The helicase of Escherichia coli, known as DnaB, is also formed from six identical protein subunits (Fass et al., 1999). DnaB is the best-characterized of the DNA helicases. It encircles the 5' lagging strand template, and translocates along it in the 5' to 3' direction. The 3' leading strand is occluded in the process. Since DnaB hexamer forms a channel wide enough for the dsDNA molecule to fit through, DnaB has the ability to mount and translocate along dsDNA without first melting it (Kaplan and O'Donnell, 2002). It can load itself onto ssDNA and translocate along it in the 5' to 3' direction until it reaches dsDNA. The helicase will melt the DNA if the substrate resembles a replication fork. Otherwise, the helicase will continue to translocate along the dsDNA molecule.

In archaea and eukaryotes, the helicases are composed of a protein called MCM. In archaeal helicases, such as that of the model organism Sulfolobus solfataricus, the structure is typically homohexameric, consisting of just one MCM protein (Brewster et al., 2008). In eukaryotes, on the other hand, MCM helicases possess six distinct subunits, Mcm2-Mcm7 (Vijayraghavan and Schwacha, 2012).

The Assembly and Activation of MCM2-7 Helicase

The assembly of helicase onto chromatin and its activation itself requires complex machinery. Consider the eukaryotic MCM2-7 helicase complex. As Takahashi et al. (2005) explain,

Briefly, the MCM2-7 complex is loaded onto origins of DNA replication in the G1 phase by at least three factors: ORC, Cdc6 and Cdt1. However, the helicase activity of the complex seems to be inactive at this stage. At the G1/S transition, at least eight more factors, including two protein kinases, are needed to activate the helicase activity of MCM2-7 and, thereby, enable origin unwinding. Among these, the initiation factors GINS and Cdc45 are particularly interesting because they are the last known proteins to be recruited before origin unwinding. In addition, both GINS and Cdc45 are required for the elongation phase of DNA replication, and both seem to exist in a physical complex with the MCM2-7 complex on chromatin. As such, Cdc45 and GINS are attractive candidates for factors that might cooperate with MCM2-7 during DNA unwinding. In support of this view, antibodies against Cdc45 block the activity of the replicative DNA helicase when it is uncoupled from the replication fork in Xenopus egg extracts. [internal citations omitted]
The Rotary Engine of DNA Helicase

A single strand of DNA passes through the central channel of the helicase hexamer, which contains DNA binding sites contributed by the helicase's subunits. A cleft in each of the subunits binds ATP via side chains in conserved residues called Sensor 1, Sensor 2, Walker A and Walker B motifs. The wave of ATP binding, hydrolysis and release, shown in the animation above, results in the DNA being passed from one subunit to the next. Together with the rotation between subunits induced by the ATP, this process causes the helicase to move forward at a rate of one nucleotide for each hydrolysis reaction.

In T7 bacteriophage, binding of a subunit to ATP causes the subunit to rotate 15 degrees (Donmez and Patel, 2006; Singleton et al., 2000).

The mechanism of helicase translocation in papillomavirus is described in a 2006 paper in Nature by Eric J. Enemark and Leemor Joshua-Tor. Since the genome of papillomavirus is circular, there are no ends available for loading of the helicase hexameric ring. Consequently, the E1 helicase has to initiate unwinding from double-stranded DNA. This is thought to be accomplished by melting the helix and loading the helicase onto a single-stranded region. This stands in contrast to the majority of DNA helicases, which need to be loaded onto a region of single-stranded DNA.

The Beta-hairpins that are present in the motor domain of the E1 helicase (and which bind DNA) form a rising staircase around the helicase's central channel. As the cycle of ATP binding, hydrolysis and release takes place, the Beta-hairpins descend the staircase. This enables the helicase to walk along the ssDNA. A similar mechanism is also present in T7 bacteriophage (Satapathy et al., 2010).

The Process of Unwinding

There are several proposed mechanisms for the unwinding of DNA by the eukaryotic MCM2-7 helicase (reviewed in Takihashi et al., 2005). These include the steric exclusion model, the rotary pump model, and the ploughshare model.

In the steric exclusion model, the helicase translocates along a single strand of the DNA molecule, thereby excluding the other strand and unwinding the duplex DNA in the process (Graham et al., 2011; Walmacq et al., 2006; Kaplan et al., 2003; Lee and Hurwitz, 2001). The rotary pump model involves the loading of the two helicases at the origins of replication. The helicases then move away from the origins before being eventually anchored and rotating in opposite directions (Laskey and Madine, 2003). According to the ploughshare model, the helicase moves along duplex DNA and uses a wedge-like protein to separate the DNA duplex as it passes through the machine, much like the cutting blade of a tractor's plough (Takihashi et al., 2005).

Conclusion

It can hardly be doubted that the DNA replication machinery is a masterpiece of nanotechnology, revealing foresight and engineering at both the macro and micro level. DNA helicase, as we have seen, reveals many characteristics of design. In future posts, I will continue to discuss elements of the DNA replication system and explore further evidence of design.


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