Which of the following enzyme separates the DNA strands

Every cell needs to divide and produce more cells in order to preserve life. The first step is to make a copy of its own DNA to pass on to daughter cells. This process of making an identical DNA is known as replication and is one of the most fundamental processes that occurs within a cell. 

The DNA present in every cell is composed of two polynucleotide strands that coil around each other to form the iconic double helix. Before DNA can be replicated, the two strands of the double helix need to be separated or ‘unzipped’. Each single strand can then be used as a template for the production of another DNA strand. This happens billions of times a day in the human body and cells need to make sure their DNA is copied very quickly, and with very few errors. To do so, they use a variety of enzymes and other proteins, which work together ensuring DNA replication is performed smoothly and accurately.

The replication is carried out by a multiprotein molecular machine known as the replisome in which three enzymes play a key role – DNA helicase, DNA polymerase and primase. Replication starts when DNA helicase binds at the replication site [origin] and moves along the DNA. While the DNA helicase is opening the double stranded DNA, primase assembles a short complementary strand of DNA, called a primer. Once the primer is in place on a single, unwound polynucleotide strand, DNA polymerase wraps itself around that strand, and attaches new nucleotides to the primer, creating a new, complementary DNA strand. One new strand [the leading strand] is made as a continuous piece, the other [the lagging strand] is made in small pieces.

The first mention of DNA helicase in the literature is from 1976 when it was discovered in E. coli and described as ATP-dependent DNA unwinding enzyme by Hoffmann‐Berling and colleagues. Two years later, the same group of scientists coined the term ‘helicase’. In the same year [1978] the first eukaryotic DNA helicase was discovered and isolated from the lily plant. Bacteriophage DNA helicase was isolated not long after [1982] the discovery of the first bacterial helicase. After that, it took almost a decade to isolate DNA helicase from human cells [1990]. Today we know that DNA helicase is a ubiquitous enzyme that plays an essential role in almost every prokaryotic and eukaryotic cell.

Interestingly, DNA helicases are ubiquitous enzymes, sequence homology reveals that most of the replication apparatus of bacterial cells evolved independently from the apparatus of eukaryotic cells.A major difference involves a swap of DNA strands used for translocation. In general, bacterial helicases translocate in the 5’-3’direction, thus prefer the lagging strand, while their eukaryotic counterparts opt for the leading strand and translocation happens in the 3’- 5’ direction.

Different DNA unwinding mechanism between bacterial and eukaryotic helicase. Bacterial helicases encircle the lagging strand and travel in the 5′–3′ direction, whereas eukaryotic helicases prefer the leading strand and travel 3′–5′.

DNA helicases are enzymes characterized by the presence of conserved motifs involved in ATP binding, ATP hydrolysis and translocation along the DNA strand. Another common feature is that they display a certain degree of amino acid sequence homology. Based on variations of the motifs and their amino acid sequence, DNA helicases are grouped into 6 superfamilies [SF]. SF1 and SF2 have at least seven conserved motifs and typically they are monomers involved in repair, recombination and transcription. On the contrary, SF3 - SF6 members assemble into hexamers involved in DNA replication. DNA helicases can also be classified as α or β depending on if they work with single or double-strand DNA [α helicases work with single-strand DNA and β helicases work with double-strand DNA]. They are also classified by translocation polarity. If translocation occurs 3’-5’ the helicase is type A, if translocation occurs 5’-3’ it is type B.

DNA repair is a crucial process in maintaining genomic integrity within a cell. Cells are constantly challenged by endogenous factors [their physiological metabolism] and/or environmental exposure [e.g. UV light, ionizing radiation] which frequently result in numerous instances of DNA damage. It is estimated that a single human cell’s DNA is damaged thousands of times every day. To fix this damage, cells have developed a system of DNA repair pathways by which they identify and correct damaged regions in affected DNA molecules, thus preventing the genomic DNA from consequences such as chromosomal instability and cancer development. Helicases have a unique place in these pathways through fork remodelling, DNA damage recognition, damaged strand removal, and recombination-based strategies.

The role of DNA helicases in replication is to encircle one strand of DNA, translocate along it and separate two strands of DNA from each other using the energy provided by the hydrolysis of a nucleoside triphosphate. This process starts when an adenine-thymine [A-T] rich sequence region is recognized and DNA helicase loads at this replication origin. The number of replication origins depends on the size of DNA. Longer DNAs have more origins and DNA replication can be initiated at all of them. It would take too long for a eukaryotic cell to replicate its genome if replication had to proceed from a single origin. Bacterial and archaeal genomes are small and usually have just a single replication origin, in contrast to eukaryotic genomes that contain significantly more origins, ranging from 400 in yeast to 30,000 – 50,000 in humans.

When the DNA helicase loads onto the origin of replication [A-T rich], it starts to “melt” the hydrogen bonds between adenine and thymine. A-T pairs are easier to separate because they have fewer hydrogen bonds between them than G-C pairs. The separation of the duplex DNA results in formation of the replication fork. The studies showed that two DNA helicases start the replication. They move in opposite directions to form two bi-directional forks in one DNA molecule. It was always believed that the two DNA helicases simply move in opposite directions from a central point. But it turned out that helicases are not facing outwards but inwards, and they need to cross each other first to leave the origin and start the DNA unzipping. 

The formation of two bi-directional forks in eukaryotic cells. When two DNA helicases pass one another and leave the origin in the 3’-5’ direction, they start producing bi-directional replication forks.

It was believed that all DNA helicases are required to be oligomeric, until the structure of a monomeric helicase complexed with a DNA was determined. This monomeric helicase [PcrA - PDB entry 3pjr], a representative member of superfamily SF1A, has been used as an inspiration for this month’s artwork. PcrA was isolated from gram-positive bacterium Geobacillus stearothermophilus and is composed of four domains termed 1A, 2A, 1B, and 2B. The crystal structure has revealed that the helicase motifs are clustered together in two RecA-like domains 1A and 2A, forming an ATP-binding pocket between them. This pocket acts like a cleftand closes in response to nucleotide binding. Along this cleft, single strand DNA binding is stabilised by stacking interactions between the bases and the side chains of aromatic residues in the acceptor pockets of the cleft. The structural data predict one nucleotide to be translocated and one base pair unwound per ATP molecule hydrolysed. 

Crystal structure of monomeric DNA helicase PcrA[PDB entry 3pjr]. Domain 1A binds on the 3’ side of the single strand DNA and domain 2A binds on 5’ side. The bound DNA triggers domain 1A to move toward domain 2A, enhancing ATP hydrolysis, resulting in translocation of PcrA helicase in the 3’ – 5’ direction along the DNA strand.

Many DNA helicases belonging to superfamilies 3-6 are hexameric and on a structural level they display a considerable similarity. They have an ATPase core with the nucleotide binding sites located at the interfaces between monomeric subunits. These nucleotide binding sites usually have an arginine finger involved in ATP binding and hydrolysis which is contributed from the neighbouring subunit. All replicative hexameric helicases also contain DNA-binding loops that extend into the central pore for DNA interaction.

Minichromosome maintenance protein [MCM] from SF6 family [PDB entry 6mii] was picked as a representative example of a hexameric DNA helicase. Six monomers assemble into a ring which encircles the DNA molecule [blue]. The ATP-binding sites are located at the interface between monomers. The neighbouring subunit provides an arginine residue [known as arginine finger] which promotes inter-subunit cooperation upon hydrolysis of ATP.

Since 1976, DNA helicase research has travelled a long way. We now know that DNA helicases are ubiquitous enzymes and are involved in almost every process in cells that concerns nucleic acid metabolism. Despite the large number of helicases that have been studied, and significant advances in our understanding of their assembly, loading, and DNA unwinding, many molecular mechanisms of their action still remain elusive.

Romana Gáborová

‘’I decided to choose the enzyme helicase, an enzyme which catalyses the separation of the two DNA strands in a helix, because I was interested in how our own emotional unravelling in the face of change correlates to that in biological systems. Helicase must separate the DNA strands to allow them to become a template for a new protein. This process not only enables genetic material to be copied and to make proteins, but also reflects our personal ‘unwinding’ or struggle in the face of growth and change. I wanted to explore how the protein helicase is a catalyst for change, like external influences in our lives that enable us to step out of our comfort zone. I used textiles to portray one strand of DNA drifting off the edge of the piece to emphasise the loss of regularity.’’

Megan Grenville from Stephen Perse Foundation in Cambridge, UK

View the artwork in the virtual 2020 PDB Art exhibition.

E. coli has 4.7 million base pairs in its genome. The time needed for their replication is on average 20-40 minutes, implying a speed of over 1000 bases per second. The typical human chromosome has about 150 million base pairs. Replication speed is 50 pairs per second. At that speed, the cell would need over a month to make a copy of a chromosome. Thanks to the presence of multiple replication origins, in fact it takes approximately only 8 hours.  

The first DNA helicase

DNA replication from two different worlds

Superfamilies

DNA replication mechanism

An unexpected mechanism

PcrA

What enzyme separates the DNA strands?

What is the separation of DNA strands called?