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Hexameric helicases some of the most complex machines on Earth

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Hexameric helicases some of the most complex machines on Earth

http://reasonandscience.heavenforum.org/t1438-hexameric-helicases-some-of-the-most-complex-machines-on-earth

The enzymes which couple chemical energy to unwind the DNA duplex are commonly referred to as helicases. Related motors also work as chromatin remodelers, which restructure chromosome organization and thereby enabling or restricting access to DNA.
The proteins that drive DNA replication—the force behind cellular growth and reproduction—are some of the most complex machines on Earth. 
DNA helicases are essential during DNA replication because they separate double-stranded DNA into single strands allowing each strand to be copied.
The helicase rotational speed of up to 10,000 rotations per minute !!!!! How astonishing and marvelous.
Helicase must have emerged before life began, since its essential for DNA replication

"The genesis of the DNA-unwinding machinery is wonderfully complex and surprising," said study coauthor Huilin Li, a biologist at Brookhaven Lab and Stony Brook University. "Seeing this helicase enzyme prepare to surround and unwind the DNA at the molecular level helps us understand the most fundamental process of life and how that process might go wrong. Errors in copying DNA are found in certain cancers, and this work could one day help develop new treatment methods that stall or break dangerous runaway machinery."

Before a cell divides
What is the driving force to make the cell divide ? Why at all did dividing start ?
it has to replicate its DNA so that the daughter cell receives a copy of the genome. The DNA helix consists of two complementary DNA strands.
Why at all did a complementary strand arise ?
Therefore, each of the two strands serves as a template for the construction of the other strand. Under normal conditions the DNA is packed into a compact structure called chromatin. To be able to replicate, the cell has to unfold and unwind the DNA,
What is the reason that it started to unwind ?
and also has to separate the two strands from each other. The cell has a complex machinery to perform these tasks.
How did this machinery arise ? What was the force behind it ? What is the origin of the information to make this machinery ?
When it is time to replicate, special initiator proteins attach to the DNA at regions called replication origins.
For what reasos did these special initiator proteins arise ? Where came the information come  from to encode these proteins ?  
These regions are characterised by a weak bond between the two DNA strands.
How did this bond arise ?
There are around 10,000 replication origins on the DNA in a cell; this arrangement increases the rate of replication tremendously. The initiator proteins pry the two strands apart
How did these proteins become able to pry the two strands apart ?
and a small gap is created at the replication origin. Once the strands are separated another group of proteins, that carry out the DNA replication, attaches and go to work.
How did these other group of proteins start to be able to attach at the right position, and go to work ?
This group of proteins includes helicase, which serves as an unzipper by breaking the bonds between the two DNA strands.
The hexameric helicases are ubiquitous proteins (related enzymes are found on both sides of the bacterial-archaean divide) involved in unwinding double-stranded DNA and RNA.
this is a highly organized, sophisticated and orchestrated movement, like a robot, executing a specific task:
In order to be able to unwind the dna strand, the initial opening of the double helix (at the origin of replication) is performed by an initiator protein.
How did chance, physical necessity, or natural selection " know " an initiator protein would be needed, and how it would have to be, and where employed ?
The helicase rotational speed of up to 10,000 rotations per minute !!!!! How astonishing and marvelous .
This unzipping takes place in both directions from the replication origins, creating a replication bubble. The replication is therefore said to be bi-directional.
How did they start and " know " the need to be bidirectional ?
Once the two strands are separated a small piece of RNA, called an RNA primer , is attached to the DNA by an enzyme called DNA primase. 4
Primases synthesize short RNA strands on single-stranded DNA templates, thereby generating the hybrid duplexes required for the initiation of synthesis by DNA polymerases. 5
These primers are the beginnings of all new DNA chains since the enzyme responsible for the copying of the DNA, DNA polymerase, can not start from scratch. It is a self-correcting enzyme and copies the DNA template with remarkable fidelity.
How did it " learn " to self-correct itself?
The DNA polymerase can only read in the 3' to 5' direction. This gives rise to some trouble since the two strands of the DNA are antiparallel. On the upper strand which runs from 3' to 5', nucleotide polymerisation can take place continuously without any problems. This strand is called the leading strand. But how does the polymerase copy the other strand then when it runs in the opposite direction, from 5' to 3'? On this so called lagging strand the polymerase produces short DNA fragments, called okazaki fragments, by using a backstitching technique.
How and why did it begin the backstitiching technique ?
These lagging strand fragments are primed by short RNA primers and are subsequently erased and replaced by DNA.



In order for DNA replication machinery to gain access to the genetic code, the two strands of the double helix must first be “unwound.” In some cases, because DNA is tightly packaged in chromatin, these protein–DNA complexes need to be restructured to expose the DNA region of interest. The enzymes which couple chemical energy to unwind the DNA duplex are commonly referred to as helicases. Related motors also work as chromatin remodelers, which restructure chromosome organization and thereby enabling or restricting access to DNA. 

The proteins that drive DNA replication—the force behind cellular growth and reproduction—are some of the most complex machines on Earth. 5 The multistep replication process involves hundreds of atomic-scale moving parts that rapidly interact and transform. Mapping that dense molecular machinery is one of the most promising and challenging frontiers in medicine and biology. "The genesis of the DNA-unwinding machinery is wonderfully complex and surprising," said study coauthor Huilin Li, a biologist at Brookhaven Lab and Stony Brook University. "Seeing this helicase enzyme prepare to surround and unwind the DNA at the molecular level helps us understand the most fundamental process of life and how that process might go wrong. But DNA replication is a bi-directional process with two helicases moving in opposite directions. The key question, then, was how does a second helicase core get recruited and loaded onto the DNA in the opposite orientation of the first?


The first DNA helicase, Escherichia coli, was purified and characterized in 1976. As more helicases were identified and reported in the literature, helicase “signature motifs” were identified. These highly conserved amino acid domains are involved in the binding and hydrolysis of nucleoside triphosphate (NTP), the energy source required to separate the stable double-stranded DNA (dsDNA). It is estimated that approximately 1% of the prokaryotic and eukaryotic genomes encode for proteins containing helicase signature motifs. In order for a helicase to unwind DNA processively, it must also be able to move along the DNA filament (i.e., to translocate) and couple this directional motion along the DNA lattice to strand separation activity.

Processive DNA unwinding requires a helicase to undergo a series of repeated “steps” along the DNA lattice until the duplex is fully unwound. Each step involves a number of processes such as 

NTP binding, 
hydrolysis, 
phosphate release, 
base pair melting or capturing of the spontaneously melted bases, and 
translocation

increasing evidence demonstrates that some helicases also possess rewinding activity—in other words, they can anneal two complementary single-stranded nucleic acids. All five members of the human RecQ helicase family, helicase PIF1, mitochondrial helicase TWINKLE, and helicase/nuclease Dna2 have been shown to possess strand-annealing activity. 4 The number of helicases expressed in higher organisms is strikingly high, with approximately 1% of the genes in many eukaryotic genomes apparently encoding RNA or DNA helicases. Helicases are involved in virtually all aspects of nucleic acid metabolism, including replication, repair, recombination, transcription, chromosome segregation, and telomere maintenance. The mechanism of this novel strand annealing activity and its biological consequences remain largely unknown



Helicase unwinds the DNA1

Helicases are enzymes that bind and may even remodel nucleic acid or nucleic acid protein complexes. There are DNA and RNA helicases. DNA helicases are essential during DNA replication because they separate double-stranded DNA into single strands allowing each strand to be copied. During DNA replication, DNA helicases unwind DNA at positions called origins where synthesis will be initiated. DNA helicase continues to unwind the DNA forming a structure called the replication fork, which is named for the forked appearance of the two strands of DNA as they are unzipped apart. The process of breaking the hydrogen bonds between the nucleotide base pairs in double-stranded DNA requires energy. To break the bonds, helicases use the energy stored in a molecule called ATP, which serves as the energy currency of cells. DNA helicases also function in other cellular processes where double-stranded DNA must be separated, including DNA repair and transcription. RNA helicases are involved in shaping the form of RNA molecules, during all processes involving RNA, such as transcription, splicing, and translation.

The hexameric helicases are ubiquitous proteins (related enzymes are found on both sides of the bacterial-archaean divide) involved in unwinding double-stranded DNA and RNA.
this is a highly organized, sophisticated and orchestrated movement, like a robot, executing a specific task:
In order to be able to unwind the dna strand,  the initial opening of the double helix (at the origin of replication) is performed by an initiator protein.
How did chance, physical necessity, or natural selection " know " an initiator protein would be needed, and how it would have to be, and where employed ?
The helicase rotational speed of up to 10,000 rotations per minute !!!!! How astonishing and marvelous.

Helicase must have emerged before life began, since its essential for DNA replication


"The genesis of the DNA-unwinding machinery is wonderfully complex and surprising," said study coauthor Huilin Li, a biologist at Brookhaven Lab and Stony Brook University. "Seeing this helicase enzyme prepare to surround and unwind the DNA at the molecular level helps us understand the most fundamental process of life and how that process might go wrong. Errors in copying DNA are found in certain cancers, and this work could one day help develop new treatment methods that stall or break dangerous runaway machinery."

How different helicase families with a conserved catalytic ‘helicase core’ evolved to function on varied RNA and DNA substrates by diverse mechanisms remains unclear. 2 Before DNA and RNA can perform their essential tasks in cells, enzymes called helicases must separate the interacting strands. A large group of helicases, known as superfamily 1 and 2, are involved in virtually all aspects of the control of RNA and DNA structure. Helicases use the energy released from breaking down molecules called nucleotides to pull apart the bonds that hold DNA and RNA strands together.

Rotary Firing in Ring-Shaped Protein Explains Unidirectionality 3

Hexameric motor proteins represent a complex class of molecular machines that variously push and pull on biological molecules using adenosine triphosphate (ATP) as chemical fuel. A specialized class of ring-shaped motor proteins, hexameric helicases, can unwind DNA strands and perform large-scale manipulations of single-stranded nucleic acids in processes such as DNA replication, DNA repair, and gene expression. To understand how certain hexameric helicases walk with directional polarity along single-stranded nucleic acids, Berkeley researchers used x-ray crystallography at the ALS to solve the structure of a hexameric helicase, the Rho transcription termination factor (from E. coli), bound to both ATP mimics and an RNA substrate. The results showed that Rho functions like a rotary engine: as the motor spins, it pulls RNA strands through it's interior. Interestingly, the rotary firing order of the motor is biased so that the Rho protein can walk in only one direction along the RNA chain.



Rings Running in Reverse

The Rho factor is a ring-shaped motor protein made up of six subunits (or, in analogy to combustion engines, six "cylinders"). Such motor proteins (also known as hexameric helicases) are found in all organisms and are involved in unwinding and moving DNA and RNA strands (nucleic acids) around the cell. There are two subfamilies of hexameric helicases: AAA+ and RecA. Rho belongs to the RecA family, which is most common in bacteria. AAA+ motors are predominantly found in eukaryotes, including humans, as well as some human pathogens, such as the papillomavirus. Although these motors evolved from a common ancestor, they have distinct properties, most notably the predisposition to walk along nucleic acid tracks in opposite directions.

To understand how such a biological mechanism works and perhaps eventually develop a therapeutic drug that will gum up the works and stop the motor from doing its job, it helps to know how the protein is constructed. Thomsen et al. are the first group to determine the crystal structure of a RecA-class hexameric helicase in a translocation state bound to both its nucleic-acid track and a molecule that mimics the role of its chemical energy source, ATP. In doing so, they fortuitously caught an atomic-level snapshot of the motor in the act of tracking along an RNA chain. Their analysis showed that the proteins from different subfamilies move in opposite directions by reversing the rotational firing order of the ATP sites—essentially by reversing gears as opposed to turning around.

Inspection of the model reveals that Rho binds to RNA in a helical conformation, using a "spiral staircase" arrangement of RNA binding loops that project from five of the protein subunits to interact with the RNA's sugar–phosphate backbone. The sixth subunit does not significantly contact the RNA and lies midway between the top and bottom steps of the staircase. The positional relationships between the six Rho subunits generate four distinct classes of ATP binding sites that together represent a complete ATP turnover cycle. The organization of these sites around the ring indicates that ATP is consumed by a sequential mechanism akin to a rotary or radial engine. The cycle of ATP binding, hydrolysis, and release is carried out as each subunit passes through the six conformational states observed in the structure, creating a rotary wave motion within the RNA binding loops to power nucleic acid translocation in the proper direction.



1. http://phys.org/news/2014-10-genesis-enzyme-dna-helix-cell.html
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383044/
3. http://www.als.lbl.gov/als/science/sci_archive/206hexhelicase.html
4. https://www.hindawi.com/journals/jna/2012/140601/
5. https://www.bnl.gov/newsroom/news.php?a=111672

More readings:
Cryo-EM structure of a helicase loading intermediate containing ORC–Cdc6–Cdt1–MCM2-7 bound to DNA
http://www.nature.com.secure.sci-hub.cc/nsmb/journal/v20/n8/full/nsmb.2629.html



Last edited by Admin on Mon Apr 24, 2017 6:41 am; edited 12 times in total

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DNA is the “blueprint of life” and stores within the necessary instructions for living cells to grow and to function. The existence of DNA has been known since 1869. It took, however, almost a century to discern DNA structure and its role in the storage of genetic information. Cellular DNA undergoes harmful modifications every day as a result of exposure to UV light, environmental stress, and toxic chemicals. DNA damage can also result from errors during DNA synthesis. Damaged DNA must be repaired promptly and efficiently; otherwise, the replication machinery can incorporate the wrong nitrogenous base, leave nicks and gaps, and stall or disengage during subsequent rounds of DNA synthesis, resulting in deleterious mutations and chromosomal instability. The cell utilizes a number of repair pathways to prevent the loss of genetic information. The enzymes that are involved in the repair process are specific to the type of DNA damage encountered and depend on the stage of the cell cycle. Not surprisingly, defects in key components of these systems in humans are associated with a broad spectrum of disorders, usually characterized by premature aging, susceptibility to cancers, and other diseases bearing hallmarks of aging, immunodeficiency, or mental retardation. Similar to DNA replication, in order for the DNA repair machinery to gain access to the genetic code, the two strands of the double helix must first be “unwound.” In some cases, because DNA is tightly packaged in chromatin, these protein–DNA complexes need to be restructured to expose the DNA region of interest. The enzymes which couple chemical energy to unwind the DNA duplex are commonly referred to as helicases. Related motors also work as chromatin remodelers, which restructure chromosome organization and thereby enabling or restricting access to DNA. 

The first DNA helicase, Escherichia coli, was purified and characterized in 1976. As more helicases were identified and reported in the literature, helicase “signature motifs” were identified. These highly conserved amino acid domains are involved in the binding and hydrolysis of nucleoside triphosphate (NTP), the energy source required to separate the stable double-stranded DNA (dsDNA). It is estimated that approximately 1% of the prokaryotic and eukaryotic genomes encode for proteins containing helicase signature motifs. In order for a helicase to unwind DNA processively, it must also be able to move along the DNA filament (i.e., to translocate) and couple this directional motion along the DNA lattice to strand separation activity.

Processive DNA unwinding requires a helicase to undergo a series of repeated “steps” along the DNA lattice until the duplex is fully unwound. Each step involves a number of processes such as

NTP binding,
hydrolysis,
phosphate release,
base pair melting or capturing of the spontaneously melted bases, and
translocation



Last edited by Admin on Sun Apr 23, 2017 2:41 pm; edited 4 times in total

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DNA Replication 1

Before a cell divides
What is the driving force to make the cell divide ? Why at all did dividing start ?
it has to replicate its DNA so that the daughter cell receives a copy of the genome. The DNA helix consists of two complementary DNA strands.
Why at all did a complementary strand arise ?
Therefore, each of the two strands serves as a template for the construction of the other strand. Under normal conditions the DNA is packed into a compact structure called chromatin. To be able to replicate, the cell has to unfold and unwind the DNA,
What is the reason that it started to unwind ?
and also has to separate the two strands from each other. The cell has a complex machinery to perform these tasks.
How did this machinery arise ? What was the force behind it ? What is the origin of the information to make this machinery ?
When it is time to replicate, special initiator proteins attach to the DNA at regions called replication origins.
For what reasos did these special initiator proteins arise ? Where came the information come  from to encode these proteins ?  
These regions are characterised by a weak bond between the two DNA strands.
How did this bond arise ?
There are around 10,000 replication origins on the DNA in a cell; this arrangement increases the rate of replication tremendously. The initiator proteins pry the two strands apart
How did these proteins become able to pry the two strands apart ?
and a small gap is created at the replication origin. Once the strands are separated another group of proteins, that carry out the DNA replication, attaches and go to work.
How did these other group of proteins start to be able to attach at the right position, and go to work ?
This group of proteins includes helicase, which serves as an unzipper by breaking the bonds between the two DNA strands.
The hexameric helicases are ubiquitous proteins (related enzymes are found on both sides of the bacterial-archaean divide) involved in unwinding double-stranded DNA and RNA.
this is a highly organized, sophisticated and orchestrated movement, like a robot, executing a specific task:
In order to be able to unwind the dna strand, the initial opening of the double helix (at the origin of replication) is performed by an initiator protein.
How did chance, physical necessity, or natural selection " know " an initiator protein would be needed, and how it would have to be, and where employed ?
The helicase rotational speed of up to 10,000 rotations per minute !!!!! How astonishing and marvelous .
This unzipping takes place in both directions from the replication origins, creating a replication bubble. The replication is therefore said to be bi-directional.
How did they start and " know " the need to be bidirectional ?
Once the two strands are separated a small piece of RNA, called an RNA primer , is attached to the DNA by an enzyme called DNA primase. 4
Primases synthesize short RNA strands on single-stranded DNA templates, thereby generating the hybrid duplexes required for the initiation of synthesis by DNA polymerases. 5
These primers are the beginnings of all new DNA chains since the enzyme responsible for the copying of the DNA, DNA polymerase, can not start from scratch. It is a self-correcting enzyme and copies the DNA template with remarkable fidelity.
How did it " learn " to self-correct itself?
The DNA polymerase can only read in the 3' to 5' direction. This gives rise to some trouble since the two strands of the DNA are antiparallel. On the upper strand which runs from 3' to 5', nucleotide polymerisation can take place continuously without any problems. This strand is called the leading strand. But how does the polymerase copy the other strand then when it runs in the opposite direction, from 5' to 3'? On this so called lagging strand the polymerase produces short DNA fragments, called okazaki fragments, by using a backstitching technique.
How and why did it begin the backstitiching technique ?
These lagging strand fragments are primed by short RNA primers and are subsequently erased and replaced by DNA.






In this youtube video its called a " fascinating protein machine ".



1. http://www.nobelprize.org/educational/medicine/dna/a/replication/
2. http://en.wikipedia.org/wiki/Helicase
3. http://www.evolutionnews.org/2013/02/unwinding_the_d_1069371.html
4. http://en.wikipedia.org/wiki/Primer_%28molecular_biology%29
5. https://jkweb.berkeley.edu/external/pdb/2000/primase/primase.html



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4 Unwinding the Double Helix: Meet DNA Helicase on Sat Nov 07, 2015 2:11 am

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Unwinding the Double Helix: Meet DNA Helicase 1

 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. AsTakahashi 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, 2006Singleton 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., 2011Walmacq et al., 2006Kaplan et al.,2003Lee 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. 


Replicating DNA with Extraordinary Fidelity: Meet DNA Polymerase 2


In a previous article, I gave a brief overview of the complex molecular mechanisms governing DNA replication. Now, I will focus specifically on the replication enzyme DNA polymerase.
DNA polymerase is the enzyme responsible for synthesizing new strands of DNA, complementary to the sequence of the template strand. The unidirectional DNA polymerase progresses along the template strand in a 3'-5' direction, since it requires a pre-existing 3'-OH group for the adding of nucleotides. The daughter strand is, consequently, synthesized in a 5'-3' direction (opposite to the direction of movement of the polymerase since the two strands have an anti-parallel orientation).
There are six different families of DNA polymerases -- A, B, C, D, X and Y (Rothwell and Waksman, 2005). These families differ from one another in their design, being specialized for a variety of purposes. For example, DNA polymerase I, found in E. coli, belongs to the A family of polymerases. Beyond its role in finishing DNA replication and removing the RNA primers, DNA polymerase I contains a 5' to 3' exonuclease domain, in addition to the 3' to 5' exonuclease domain, that allows it to remove nucleotides both in front of and behind it (more on proofreading by exonuclease domains shortly) (Ishino et al., 1995). Polymerases in the B, C and D families are known for their high fidelity (owing to their intrinsic 3' to 5' proofreading exonuclease), and are found in eukaryotes, bacteria and archaea respectively. The X family (e.g. eukaryotic polymerases pol β, Pol σ, Pol λ, Pol μ) plays a role in DNA repair, filling in the gaps created during the process (Yamtich and Sweasy, 2010). Whereas most polymerases cannot replicate past bulky lesions in damaged DNA, the Y family are able to replicate past them (Washington et al., 2010).
DNA polymerases generally share a common structural framework, with fingers, thumb and palm subdomains that make up the polymerase domain. The diagram below, excerpted from Beard and Wilson (2003), shows the structure of T7 DNA polymerase, revealing its two domains, a polymerase domain (colored) and a proofreading exonuclease domain (gray). The polymerase domain is comprised of three subdomains: fingers, thumb and palm. The finger domain positions incoming nucleoside triphosphates in relation to the template strand. The thumb domain is thought to function in the processivity, positioning and translocation of the DNA, holding in place the elongating DNA duplex. The β-sheet that comprises the palm domain is where the enzyme's active site lies, which catalyzes the transfer of phosphoryl groups in the phosphoryl transfer reaction.

How does DNA polymerase add new nucleotides to the elongating strand? The polymerase's active site, found in the β-sheet that makes up the palm subdomain catalyes a phosphoryl transfer reaction. It forms a phosphodiester bond by linking the 3' hydroxyl group at the end of the template strand to the nucleotide's 5' phosphoryl group. The first step in the process is a nucleophilic attack on the α-phosphate of the incoming nucleoside triphosphate by the 3' OH of the growing chain. This reaction releases pyrophosphate (PPi). Within the active site, there are two conserved aspartate residues. The magnesium ions on the carboxylate groups of those aspartates is critical to the reaction. These carboxylate groups co-ordinate the magnesium ions and facilitate their participation in the catalysis by holding them in the right orientation. One of the two magnesium ions activates the 3' OH group of the terminal nucleotide. The other is responsible for stabilizing a developing negative charge on the leaving oxygen on the incoming nucleoside triphosphate. Side chains on an alpha helix in the finger domain interact with the incoming triphosphate to also stabilize it. Hydrolysis of the pyrophosphate released in this process generates the energy required for driving the reaction forward. For a more detailed review of the mechanisms involved, I refer readers to Rothwell and Waksman (2005).
The process by which DNA polymerase selects the correct nucleotide is less well understood. For a discussion, I refer readers to Markiewicz et al. (2012).
As I stated in my previous post, the rate at which DNA polymerase replicates DNA is thought to be a whopping 749 nucleotides per second (McCarthy et al., 1976) and the error rate for accurate polymerases is believed to be in the range of 10^-7 and 10^-8, based on studies of E. coli and bacteriophage DNA replication (Schaaper, 1993). This extraordinarily high fidelity is accomplished by a remarkable proofreading and error-correcting facility built into the enzyme, which checks the identity of nucleotides both during and after polymerization.


The first level of monitoring occurs by virtue of the fact that, when base-paired with the complementary strand (A with T, or C with G), correct nucleotides precisely fit into the active site, whereas nucleotides that are incorrectly matched will possess a different geometry and will not will not fit so precisely into the active site (Johnson and Beese, 2004).
Sometimes, however, this first level of monitoring will fail to prevent the entry of an incorrect nucleotide. But, thankfully, there is also a second level of proofreading. In addition to the polymerase active site, DNA polymerase possesses a 3' to 5' exonuclease active site, which can cleave an incorrect nucleotide from the 3' end of the growing DNA strand before synthesis of the subsequent nucleotide. When an incorrect nucleotide is mistakenly incorporated, the polymerase's rate of activity is significantly delayed. Studies have shown that the presence of mismatch can reduce the polymerase's efficiency of subsequent elongation by as much as a hundred to a million fold (Kunkel and Bebenek, 2000Goodman et al., 1993Echols and Goodman, 1991). This gives enough time for spontaneous denaturing of the DNA at the 3' end, thereby facilitating the transfer of the 3' end with the mismatched nucleotide to the polymerase's 3' exonuclease site, which catalyzes the removal of multiple nucleotides from the 3' end of the DNA strand. The 3' end is subsequently positioned back into the polymerase active site, and the polymerase can then continue its DNA synthetic activity in the 5' to 3' direction.
The following animation reveals this remarkable process at work:






It should be noted, however, that not all DNA polymerases possess an intrinsic proofreading exonuclease. Polymerases belonging to the Y family, for example, tend to be significantly less accurate (Friedberg et al., 2001). Members of this family "lack an intrinsic proofreading exonuclease, exhibit low processivity, replicate DNA with low fidelity, and are believed to assist replication complexes stalled at DNA lesions," (Beard et al., 2002).
DNA polymerase is just one of numerous protein complexes that play an important role in DNA replication. A general overview of the machinery involved in the process of replication is more than adequate grounds to justify a design inference. As we drill down and examine the individual subcomponents that make up the cell's DNA replication machinery, the case for design becomes ever harder to ignore. In subsequent articles I will continue this exploration of the intricate molecular processes underlying DNA biosynthesis.



1) http://www.evolutionnews.org/2013/02/unwinding_the_d_1069371.html
2) http://www.evolutionnews.org/2013/01/replicating_dna068131.html

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