Introduction

Deoxyribonucleic acid (DNA) is a nucleic acid that is made up of three components: a deoxyribose sugar, a phosphate, and a nitrogenous base. Deoxyribonucleic acid, DNA is the genetic material via which a cell is defined. It is a long molecule containing unique codes that give instructions for the synthesis of all body proteins.

DNA structure

• The structural model of DNA was initially proposed by James Watson and Francis Click.
• They found that DNA is a double-helical structure with two paired DNA strands with complementary nucleotide sequences.
• The double-stranded DNA molecule has two spiral nucleic acid chains that are twisted into a double helix shape. The twisting gives the DNA its compactness.
• DNA is made up of millions of nucleotides. Nucleotides are molecules that are composed of deoxyribose sugar, with a phosphate group and a nucleobase that is attached to it.
• Each nucleotide is tightly base paired with a complementary nucleotide on the opposite strand, i.e Adenine (A) paired with Thymine (T) or Guanine (G) paired with cytosine (C), and therefore one strand’s sequence acts as a template for the new strand to be formed during replication.
• Nucleotides are bound to each other in strands via phosphodiester bonds forming a sugar-phosphate backbone.
• They form a bond that is between the third carbon atom on the deoxyribose sugar made up of one sugar thus it is designated as the 3′ (three prime) and the fifth carbon atom of another sugar on the next nucleotide as the 5′ (five prime).
• Any part of the sequence can be used to create or recognize its adjacent nucleotide sequence during replication.
• DNA fits within the nucleus by being closely packed into tight coils known as chromatins. The chromatins condense to form the chromosomes during cell division.
• Before DNA replication, the chromatins loosen up giving the replication machinery access to the DNA strands.

What is DNA Replication?

• This is a complex process that takes place during cell division, (interphase, S phase) whereby DNA makes copies (duplicates) before the cell divides through mitosis and meiosis.
• DNA replication is a semiconservative process where a parental strand (template) is used to synthesize a new complementary daughter strand using several protein elements which include enzymes and RNA molecules.
• DNA replication process uses DNA polymerase as the main enzyme for catalyzing the joining of deoxyribonucleoside 5′-triphosphates (dNTPs) forming a growing chain of DNA.
• Other proteins are also involved for initiation of the process and copying of DNA, along with proofreading capabilities to ensure the replication process takes place accurately.
• Therefore DNA replication is a process that produces identical helices of DNA from a single strand of the DNA molecule.
• DNA replication is an essential mechanism in enhancing cell growth, repair, and reproduction of an organism.

Figure: The mechanism of DNA replication. Image Source: MBInfo © 2018 National University of Singapore.

The mechanism of DNA replication

Summary: DNA replication takes place in three major steps.

1. Opening of the double-stranded helical structure of DNA and separation of the strands
2. Priming of the template strands
3. Assembly of the newly formed DNA segments.

• During the separation of DNA, the two strands uncoil at a specific site known as the origin. With the involvement of several enzymes and proteins, they prepare (prime) the strands for duplication.
• At the end of the process, DNA polymerase enzyme starts to organize the assembly of the new DNA strands.
• These are the general steps of DNA replication for all cells but they may vary specifically, depending on the organism and cell type.
• Enzymes play a major role in DNA replication because they catalyze several important stages of the entire process.
• DNA replication is one of the most essential mechanisms of a cell’s function and therefore intensive research has been done to understand its processes.
• The mechanism of DNA replication is well understood in Escherichia coli, which is also similar to that in eukaryotic cells.
• In E.coli, DNA replication is initiated at the oriClocus (oriC), to which DnaA protein binds while hydrolyzing of ATP takes place.

DNA replication enzymes and Proteins

DNA polymerase

• DNA polymerases are enzymes used for the synthesis of DNA by adding nucleotide one by one to the growing DNA chain. The enzyme incorporates complementary amino acids to the template strand.
• DNA polymerase is found in both prokaryotic and eukaryotic cells. They both contain several different DNA polymerases responsible for different functions in DNA replication and DNA repair mechanisms.

DNA Helicase enzyme

• This is the enzyme that is involved in unwinding the double-helical structure of DNA allowing DNA replication to commence.
• It uses energy that is released during ATP hydrolysis, to break the hydrogen bond between the DNA bases and separate the strands.
• This forms two replication forks on each separated strand opening up in opposite directions.
• At each replication fork, the parental DNA strand must unwind exposing new sections of single-stranded templates.
• The helicase enzyme accurately unwinds the strands while maintaining the topography on the DNA molecule.

DNA primase enzyme

• This is a type of RNA polymerase enzyme that is used to synthesize or generate RNA primers, which are short RNA molecules that act as templates for the initiation of DNA replication.

DNA ligase enzyme

• This is the enzyme that joins DNA fragments together by forming phosphodiester bonds between nucleotides.

Exonuclease

• These are a group of enzymes that remove nucleotide bases from the end of a DNA chain.

Topoisomerase

• This is the enzyme that solves the problem of the topological stress caused during unwinding.
• They cut one or both strands of the DNA allowing the strand to move around each other to release tension before it rejoins the ends.
• And therefore, the enzyme catalysts the reversible breakage it causes by joining the broken strands.
• Topoisomerase is also known as DNA gyrase in E. coli.

Telomerase

• This is an enzyme found in eukaryotic cells that adds a specific sequence of DNA to the telomeres of chromosomes after they divide, stabilizing the chromosomes over time.

Video: DNA replication enzymes and their functions (Shomu’s Biology)

DNA Replication Steps/Stages

Initiation

• This is the stage where DNA replication is initiated.
• DNA synthesis is initiated within the template strand at a specific coding region site known as origins.
• The origin sites are targeted by the initiator proteins, which recruit additional proteins that help in the replication process to form a replication complex around the DNA origin.
• There are several origin sites on which DNA replication is initiated and they are all known as replication forks.
• The formed replication complex contains the DNA helicase enzyme whose function is to unwind the double helix, exposing the two strands, which act as templates for replication.
• The mechanism of DNA helicase enzyme is by hydrolyzing the ATP that is used to form the bonds between the nucleobases, thus breaking the bond that holds the two strands.
• Additionally, during initiation DNA primase enzyme synthesizes small RNA primers that kick-start the function of DNA polymerase.
• DNA polymerase enzyme functions by growing the new DNA daughter strand.

Elongation

• This is the phase where the DNA polymerase grows the new DNA daughter strand by attaching to the original unzipped template strand and the initiating short RNA primer.
• The DNA polymerase is able to synthesize a new strand that matches the template, by extending the primer via the addition of free nucleotides to the 3′ end.
• One of the templates reads in the 3′ to 5′ direction, and therefore, the DNA polymerase synthesizes the new strand in the 5′ to 3′ direction, which is known as the leading strand.
• Along the template strand, DNA primase synthesizes a short RNA primer at the beginning of the template in the 5′ to 3′ direction, which initiates the DNA polymerase to continue synthesizing new nucleotides, extending the new DNA strand.
• The other template (5′ to 3′) is elongated in an antiparallel direction, by the addition of short RNA primers which are filled with other joining fragments, forming the newly formed lagging strand. These short fragments are known as the Okazaki fragments.
• The synthesis of the lagging strand is discontinuous since the newly formed strand is disjointed.
• The RNA nucleotides from the short RNA primers must be removed and replaced by DNA nucleotides, which are then joined by the DNA ligase enzyme.

Termination

• After the synthesis and extension of both the continuous and discontinued stands, an enzyme knows as exonuclease removes all RNA primers from the original strands.
• The primers are replaced with the right nucleotide bases.
• While removing the primers, another type of exonuclease proofread the new stands, checking, removing, and replacing any errors formed during synthesis.
• DNA ligase enzyme joins the Okazaki fragments to form a single unified strand.
• The ends of the parent strand consist of a repetition of DNA sequences known as telomeres which act as protective caps at the ends of chromosomes preventing the fusion of nearby chromosomes.
• The telomeres are synthesized by a special type of DNA polymerase enzyme known as telomerase.
• It catalyzes the telomere sequences at the end of the DNA.
• On completion, the parent and complementary strand coil into a double helical shape, producing two DNA molecules each passing one strand from the parent molecule and one new strand.

DNA Replication Video Animation (Amoeba Sisters)

Okazaki fragments

• The two DNA strands run in opposite or antiparallel directions, and therefore to continuously synthesize the two new strands at the replication fork requires that one strand is synthesized in the 5’to3′ direction while the other is synthesized in the opposite direction, 3’to 5′.
• However, DNA polymerase can only catalyze the polymerization of the dNTPs only in the 5’to 3’direction.
• This means that the other opposite new strand is synthesized differently. But how?
• By the joining of discontinuous small pieces of DNA that are synthesized backward from the direction of movements of the replication fork. These small pieces or fragments of the new DNA strand are known as the Okasaki Fragments.
• The Okasaki fragments are then joined by the action of DNA ligase, which forms an intact new DNA strand known as the lagging strand.
• The lagging phase is not synthesized by the primer that initiates the synthesis of the leading strand.
• Instead, a short fragment of RNA serves as a primer (RNA primer) for the initiation of replication of the lagging strand.
• RNA primers are formed during the synthesis of RNA which is initiated de novo, and an enzyme known as primase synthesizes these short fragments of RNA, which are 3-10 nucleotides long and complementary to the lagging strand template at the replication fork.
• The Okazaki fragments are then synthesized by the extension of the RNA primers by DNA polymerase.
• However, the newly synthesized lagging strand is that it contains an RNA-DNA joint, defining the critical role of RNA in DNA replication.

Figure: Okazaki fragments. Image Source: David O Morgan.

Replication Fork Formation and its function

• The replication fork is the site of active DNA synthesis, where the DNA helix unwinds and single strands of the DNA replicates.
• Several sites of origin represent the replication forks.
• The replication fork is formed during DNA strand unwinding by the helicase enzyme which exposes the origin of replication. A short RNA primer is synthesized by primase and elongation done by DNA polymerase.
• The replication fork moves in the direction of the new strand synthesis. The new DNA strands are synthesized in two orientations, i.e 3′ to 5′ direction which is the leading strand, and the 5′ to 3′ orientation which is the lagging strand.
• The two sides of the new DNA strand (leading and lagging strand) are replicated in two opposite directions from the replication fork.
• Therefore the replication fork is bi-directional.

Figure: DNA Replication Fork. Image Source: MDPI (Adam R. Leman and Eishi Noguchi).

Leading Strand

• The leading strand is the new DNA strand that is continuously synthesized by the DNA polymerase enzyme.
• It is the simplest strand that is synthesized during replication.
• The synthesis starts after the DNA strand has unzipped and separated. This generates a short piece of RNA known as a primer, by the DNA primase enzyme.
• The primer binds to the 3′ end (start) of the strand, thus initiating the synthesize of the new strand (leading strand).
• The synthesis of the leading strand is a continuous process.

The Lagging Strand

• This is the template strand (5′ to 3′) that is synthesized in a discontinuous manner by RNA primers.
• During the synthesis of the leading strand, it exposes small, short strands, or templates that are then used for the synthesis of the Okasaki fragments.
• The Okasaki fragments synthesize the lagging strand by the activity of DNA polymerase which adds the pieces of DNA (the Okasaki fragments) to the strand between the primers.
• The formation of the lagging strand is a discontinuous process because the newly formed strand (lagging strand) is the fragmentation of short DNA strands. 

Why is DNA replication important?

• DNA replication takes place during cell division and it enables the multiplication and division of DNA by making two copies of the genome from a single parent genome.
• And therefore, its importance is in the creation of new and next copies of DNA giving rise to two daughter cells from a single parent cell.
• Each new cell is formed with its own genome.
• This enhances heredity via reproduction and cell division.

DNA replication stress

During DNA replication, the process and the DNA genome undergoes various stress arising from the mechanism. these stresses an result in stalled replication and stalled replication fork formation. Several events contribute to these stresses, including;

• Unusual DNA structure
• Mismatched ribonucleotides
• Tensions arising from concurrent mechanisms of replication and transcription
• Inadequate availability of important replication factors
• Fragile sites on the replicating DNA strand
• Overexpression or constitutive activation of oncogenes
• Inaccessible chromatins

Kinase regulatory proteins such as ATM (ATM serine/threonine kinase) and ATP are proteins that assist in alleviating replication stress. These proteins get recruited and activated by DNA damages.

Stalled replication forks may collapse if the regulatory proteins do not stabilize, and if and when this happens, initiation of repairing mechanisms to reassembling of the replication fork takes place. this helps to amend damages the damaged ends of DNA.

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DNA Replication in Eukaryotes (Differences with prokaryotes)

DNA replication in prokaryotes and eukaryotes have several similar features and also differences. This depends on the cell sizes and genome sizes.

Similarities between Prokaryotic and Eukaryotic DNA Replication

• The unwinding mechanism of DNA before replication is initiated is the same for both Prokaryotes and eukaryotes.
• In both organisms, the DNA polymerase enzyme coordinated the synthesis of new DNA strands.
• Additionally, both organisms use the semi-conservative replication pattern, making the leading and lagging strands in different directions. Okasaki fragments make the lagging strand.
• Lastly, both organisms initiate DNA replication using a short RNA primer.

Differences between DNA replication in Eukaryotes and Prokaryotes