Definition of DNA
The genetic instructions for every individual on Earth are stored in a very long molecule called DNA, or deoxyribonucleic acid. It contains the blueprints for building all the proteins in our body, much like a cookbook.
- DNA, or deoxyribonucleic acid, is the molecule that stores the instructions for building and maintaining an organism.
- DNA is a double helix, a structure consisting of two helical strands that are connected together and spiral around one other.
- The backbone of each strand is a chain of sugar (deoxyribose) and phosphate groups that alternates along the length of the strand.
- One of four bases—adenine (A), cytosine (C), guanine (G), or thymine (T)—is linked to each sugar (T).
- Bases form chemical connections with one another to connect the two strands; adenine binds to thymine, and cytosine to guanine.
- Biological information, like the blueprint for a protein or RNA molecule, is stored in the order of the bases along DNA’s backbone.
- During DNA replication, the strands split.
- Francis Crick, James Watson, Rosalind Franklin, and Maurice Wilkins were the original discoverers of the DNA double helix structure.
- Although the human genome has 3.2 billion bases of DNA, the genomes of other creatures can range from a few hundred million to billions of bases.
Structure of DNA
- We may compare the DNA structure to a twisted ladder.
- According to the definition, its shape resembles a double helix, as seen in the diagram.
- A nucleic acid, because all nucleic acids consist of nucleotides.
- Each nucleotide in the DNA molecule consists of three separate parts, including a sugar, a phosphate group, and a nitrogen base.
- Nucleotides consist of a sugar moiety, a phosphate moiety, and a nitrogen base, and they are the fundamental units of DNA.
- Each strand of DNA is made up of nucleotides, which are bonded together by sugar and phosphate groups.
- There are four different nitrogen bases, which are designated by the letters A, T, G, and C, respectively.
- The following are the possible combinations of the four nitrogenous bases: A with T, and C with G.
- These nucleotide pairs are fundamental to maintaining the DNA’s double helix shape (which looks like a twisted ladder).
- The genetic code, or DNA instructions, are determined by the sequence of nitrogenous bases.
- Sugar is the structural backbone of the DNA molecule together with phosphorus and nitrogen. It’s also known as deoxyribose.
- By creating hydrogen bonds, the nitrogenous bases of complementary strands are able to arrange themselves in a ladder pattern.
- In order to make a nucleotide, the DNA molecule requires four nitrogen bases: adenine (A), thymine (T), cytosine (C), and guanine (G).
- Purines are A and G, whereas pyrimidines are C and T.
- DNA has two strands, and they go in opposing directions.
- The presence of a hydrogen bond between the two complimentary bases helps to keep these strands together.
- Each strand forms a right-handed coil, and a turn of the helix consists of 10 nucleotides.
- 3.4 nm is the pitch of each helix.
- Thus, 0.34 nm is the distance between two successive base pairs (i.e., hydrogen-bonded bases of opposing strands).
- There is only one DNA molecule in each chromosome that coils up to create the chromosome.
- About 23 sets of chromosomes are found in each human cell nucleus.
- Cell division is another crucial function that relies heavily on DNA.
What is DNA Replication?
DNA replication occurs during cell division and involves the creation of an exact copy of the original DNA. When DNA replicates, it creates many copies of itself.
- It’s a form of biological polymerization that involves a chain of steps that begins with initiation and ends with termination.
- This transformation is facilitated by enzymes.
- The key enzyme in this process is called DNA Polymerase.
- Due to the semiconservative nature of DNA replication, each strand of the DNA double helix is used as a guide in the creation of a new, complimentary strand.
- From a single parent molecule, we get two “daughter” molecules having one new and one old strand in each double helix.
- Many different proteins and enzymes are required for the process of DNA replication. The enzyme DNA polymerase (also known as DNA pol) plays a pivotal role. DNA pol I, DNA pol II, and DNA pol III are the three most common DNA polymerases found in bacteria.
- DNA polymerase III (pol III) is the enzyme needed for new DNA to be created, while DNA polymerase I (pol I) and DNA polymerase II (pol II) are mostly needed for DNA repair. DNA polymerase III (DNA pol III) is responsible for adding to the 3′-OH group of the developing DNA chain deoxyribonucleotides that are corresponding to nucleotides on the template strand.
- DNA replication is a multi-step process that requires a wide variety of enzymes and other proteins due to the complexity of eukaryotic genomes. Initiation, development, and completion are the three major phases of this process.
- DNA, or deoxyribonucleic acid, is a kind of nucleic acid that consists of the sugar deoxyribose, the phosphate, and the nitrogenous base.
- When a cell splits into two daughter cells, it must copy the DNA in order to preserve the organism’s genetic information. Replication refers to the process through which DNA is duplicated.
- DNA helices are replicated from a single double-stranded molecule during replication.
- Enzymes play a crucial role in DNA replication by catalysing critical reactions.
- Both cell expansion and reproduction rely heavily on the DNA replication mechanism. Further, it plays a crucial role in mending damaged cells.
Mechanism of dna replication/Principle of DNA replication
There are three main processes involved in DNA replication.
1. DNA strands unwind from their double-helix shape and separate.
2. Template-strand priming
3. Putting together the pieces of DNA that were just created.
- DNA unwinds into two strands from a single molecule at a location called the origin of replication. Several enzymes and proteins work together to “prime” DNA strands so that they are ready to be duplicated.
- DNA polymerase enzyme then begins to coordinate the building of the new DNA strands at the conclusion of the process.
- DNA replication follows these broad processes in every cell, while the details may change depending on the organism and the kind of cell.
- DNA replication relies heavily on enzymes since they catalyse numerous crucial steps.
- DNA replication is a fundamental cellular operation, therefore scientists have spent a lot of time trying to figure out how it works.
- DNA replication in Escherichia coli is quite similar to DNA replication in eukaryotic organisms, and its process is therefore well understood.
- DnaA protein interacts to the oriClocus (oriC) in E. coli, where DNA replication begins in tandem with ATP hydrolysis.
Requirement for DNA replication – Enzymes and Proteins
Protein enzymes play a crucial role in the replication of DNA. Numerous enzymes, such as DNA-dependent DNA polymerase, helicase, ligase, etc., are required for the replication of DNA. However, DNA-dependent DNA polymerase is the most important enzyme of all of them.
1. DNA-dependent DNA polymerase
- Together with other enzymes, it aids in the polymerization, catalyses, and regularises the entire process of DNA replication.
- Deoxyribonucleoside triphosphates serve as both the substrate and the energy source for the replication process. There are 3 distinct forms of DNA polymerase:
a. DNA Polymerase I
The enzyme DNA Polymerase I is used to repair DNA. It’s engaged in three pursuits:
- 5′-3′ polymerase activity
- 5′-3′ exonuclease activity
- 3′-5′ exonuclease activity
b. DNA Polymerase II
- Primer extension and proofreading are two of DNA Polymerase II’s many duties.
c. DNA Polymerase III
- It catalyzes DNA replication in living cells as DNA Polymerase III.
2. DNA helicase (DnaB Protein)
- Protein helicase that unwinds DNA (DnaB Protein)
- DNA helicase is a protein that travels along DNA and unwinds and separates double-stranded DNA.
- By severing DNA’s hydrogen bonds, it can produce the replication fork.
3. DNA primase (DnaG Protein)
- DNA primase is an RNA polymerase that acts as an RNA primer synthesiser.
- DNA replication starts with the help of primers, which are little RNA molecules that serve as guides.
4. Topoisomerase or DNA Gyrase
- To keep DNA from being knotted up or supercoiled, enzymes called topoisomerases or DNA gyrases unwind and rewind the strands.
- In DNA, the last few nucleotides are removed by a class of enzymes called exonucleases.
6. DNA ligase
- DNA ligase is an enzyme that forms phosphodiester linkages between nucleotides, therefore joining DNA fragments.
7. Single-stranded Binding Proteins
- It prevents secondary structures from developing by binding to single-stranded DNA.
- Eukaryotic cells have an enzyme called telomerase, which helps stabilise chromosomes by adding a sequence of DNA to the ends of chromosomes each time they split.
9. Tus Protein
- Tus protein is involved in the last stages of replication.
10. SSB Protein
- After the DNAs have been split by helicase, bind to the single stranded DNA.
11. DnaA Protein
- DnaA is a protein that attaches to the replication origin and is essential for the process to begin.
12. Helicase loader (Dna C Protein)
- Help guide the helicase to the DNA template with the help of DnaA.
DNA Replication Steps/Stages
- Here, the process of copying DNA begins.
- DNA synthesis begins at designated sites in the template strand’s coding regions called origins.
- Initiator proteins specifically seek for origin sites, where they then assemble a replication complex at the DNA’s starting point.
- There are many places where DNA replication starts, and these starting points are all called replication forks.
- It is the job of the DNA helicase enzyme, which is part of the replication complex, to unwind the double helix and expose the two strands that serve as replication templates.
- DNA helicase enzymes work by hydrolyzing the ATP needed to create the connections between nucleobases, releasing the strands from their mutual holding pattern.
- DNA polymerase is activated by tiny RNA primers, which are synthesised by the DNA primase enzyme during initiation.
- DNA polymerase is an enzyme whose job it is to create a new strand of DNA.
- This is when the DNA polymerase grows the new DNA daughter strand by joining the unzipped template strand with the short RNA primer.
- By adding free nucleotides to the 3′ end of the primer, DNA polymerase is able to synthesis a new strand that is complementary to the template strand.
- Since one of the templates is read from 3′ to 5′, DNA polymerase must manufacture the new strand from 5′ to 3′, thus the term “leading strand.”
- DNA primase synthesises a short RNA primer at the 5′ end of the template in the 3′ to 5′ direction, activating DNA polymerase to continue synthesising new nucleotides and elongating the new DNA strand.
- The trailing strand is created by adding short RNA primers, which are filled with additional joining pieces, to the other template, which is lengthened in an antiparallel way (5′ to 3′). The Okazaki fragments are a collection of such snippets.
- Since the freshly produced strand is incomplete, the synthesis of the trailing strand is also incomplete.
- DNA ligase is an enzyme that joins DNA strands together after the RNA nucleotides in short RNA primers are removed.
- Exonuclease is an enzyme that eliminates all RNA primers from the original strands after synthesis and extension of both the continuous and discontinued strands.
- Correct nucleotide bases are inserted into the primers.
- Another kind of exonuclease checked the new stands for faults made during synthesis when the primers were being removed.
- The Okazaki fragments are reassembled by the DNA ligase enzyme.
- Telomeres, which are found at the ends of chromosomes, are a protective cap that prevents the fusion of neighbouring chromosomes by repeating a specific sequence of DNA.
- One enzyme, telomerase, is responsible for synthesising telomeres, which are the ends of chromosomes.
- It activates DNA segments known as telomeres.
- When the process is finished, two new DNA molecules are formed by passing one strand from the parent molecule through the newly formed helix on the complementary strand.
What are Okazaki fragments?
- The two DNA strands run in opposing, or antiparallel, orientations, therefore in order to continually synthesis the two new strands at the replication fork, one strand must be generated in the 5’to3′ direction while the other is formed in the 3’to5′ way.
- DNA polymerase can only catalyse the polymerization of the dNTPs in the 5′-to-3′ orientation.
- In other words, the synthesis of the new strand on the other side of the molecule is different. Sure, but how?
- By fusing together short, broken strands of DNA generated in the opposite direction of the replication fork. Okasaki fragments refer to the new DNA strand broken up into smaller bits.
- DNA ligase subsequently joins the Okasaki pieces to create a single, unbroken strand of DNA, which is called the lagging strand.
- Unlike the leading strand, which is formed by the primer, the lagging strand is not created by this process.
- Instead, the replication of the lagging strand is kicked off by using a little piece of RNA as a primer (RNA primer).
- Primers are small RNA pieces (about 3-10 nucleotides in length) that are complementary to the lagging strand template at the replication fork and are produced during the initiation of RNA synthesis via de novo transcription.
- DNA polymerase uses RNA primers as a template to create Okazaki fragments.
- The RNA-DNA junction seen in the freshly generated lagging strand, however, defines the important role of RNA in DNA replication.
Read Also: MCQ questions on DNA Replication
What is Replication Fork?
- As the DNA helix unwinds at the replication fork, individual DNA strands are copied.
- The replication forks can be thought of as having several starting points.
- DNA strand unwinding by the helicase enzyme reveals the replication origin and results in the formation of the replication fork. Primase is responsible for synthesising a small RNA primer, and DNA polymerase is responsible for elongation.
- When a new strand of DNA needs to be synthesised, the replication fork will head in that direction. Both the leading strand, which is formed in a 3′ to 5′ direction, and the lagging strand, which is synthesised in a 5′ to 3′ manner, are produced during DNA replication.
- The replication fork splits in two, allowing either half of the new DNA strand (the leading strand and the lagging strand, respectively) to duplicate in opposite directions.
- This means that replication can proceed in either way at the bifurcation.
- Fork formation during DNA replication
- The DNA polymerase enzyme constantly creates a new strand of DNA, and this strand is known as the leading strand.
- During replication, the simplest strand is generated.
- Once the two ends of the DNA strand have unwound, synthesis can begin. By doing so, the DNA primase enzyme creates a little snippet of RNA used as a starting point for DNA replication.
- A new strand of DNA is synthesised once a primer binds to its 3′ end (its beginning) (leading strand).
- Synthesis of the leading strand occurs in a continuous manner.
The Lagging Strand
- Disjointedly, RNA primers synthesise the template strand (5′ to 3′).
- When the leading strand is being synthesised, short strands are exposed and employed as templates to create the Okasaki fragments.
- DNA polymerase is responsible for synthesising the lagging strand from the Okasaki fragments, which are added to the strand between the primers.
- Since the newly created strand (lagging strand) is the fragmentation of short DNA strands, the development of the lagging strand is a discontinuous process.
DNA Replication Process in Prokaryotes
Within prokaryotes, DNA replication occurs at:
- At the site where DNA replicates, the two strands of the molecule separate from each other.
- Replication forks can occur after the DNA has been opened by helicase.
- Single-strand binding proteins wrap the DNA around the replication fork to keep it from unwinding.
- DNA supercoiling can be avoided with the help of topoisomerase.
- Primase is a protein responsible for the synthesis of RNA primers. The DNA strand and these primers are a perfect match.
- Initiation of nucleotide addition by DNA polymerase III occurs at the primer 3′-end.
- Both the fore and aft strands are getting longer.
- After the primers are eliminated, DNA Polymerase I is used to fill in the gaps, and ligase is used to glue the resulting strands together.
DNA Replication in Eukaryotes
Replication of DNA in eukaryotes is quite similar to that in prokaryotes. Yet eukaryotes’ starting procedure is more intricate than that of prokaryotes. Many independent sites of replication initiation exist in eukaryotic cells. Together with other initiator proteins, it forms a pre-replication complex. Despite using different enzymes, the method is otherwise same. The enzyme Pol δ is responsible for polymerization in eukaryotes, whereas DNA Pol III is responsible for this activity in prokaryotes.
DNA Replication Summary
During the process of cell division, DNA replication plays a crucial role. DNA makes a copy of itself in this process, which is also known as semi-conservative replication.
There are three steps involved in the replication of DNA:
Step 1: Initiation
At a site called the origin of replication, DNA copies are made. DNA helicase functions by unwinding double helix of DNA. The replication fork is thus created.
Step 2: Elongation
DNA polymerase III copies the template strand’s nucleotides by adding complimentary nucleotides in a sequential fashion. If it detects an adenine in the template strand, for instance, it will substitute a thymine in its place.
Because of the process of adding nucleotides to the lagging strand, spaces are created in between the strands. Okazaki gaps refer to such shattered pieces of information. Ligase is used to repair these holes or slashes.
Step 3: Termination
The replication process is stopped when a termination sequence is located opposite the replication origin. The DNA polymerase chain reaction is stopped when the TUS protein (terminus utilisation substance) attaches to the terminator sequence. In other words, it causes death.
DNA is the hereditary material in which all genetic information is stored. Most genes have between two hundred and two million base pairs (base pairs) in length. In the genetic code for polypeptides, each amino acid is represented by a sequence of three nitrogenous bases.
Proteins are formed when polypeptide chains are folded into secondary, tertiary, and quaternary structures. Different proteins can be made since there are many different genes in the DNA of every living thing. Proteins serve as the primary molecular structural and functional units in most living creatures. DNA plays a role in, besides storing genetic information,
- The process of replication involves the uniform dispersion of DNA during cell division as well as the transmission of genetic information from a mother cell to her daughter cells and from one generation to the next.
- Mutations: DNA sequence variations
- Processes Within Cells
- DNA Tracing Gene Therapy Through a Patient’s DNA
Importance of DNA replication
- DNA stores the instructions for making proteins and enzymes that are essential for a cell to function.
- When a cell divides, one copy of its DNA is copied and transmitted to the daughter cell.
- Serious problems can arise if DNA replication fails because the resulting daughter cell lacks the machinery necessary to synthesise proteins.
- In autosomal cells, the diploid condition is kept due to DNA replication.
- Genetic information is passed down from one generation to the next via changes in DNA. The development of subtle alterations occurs during DNA replication. Inadvertent changes like this cause mutations. Evolutionary theory and biological diversity both have their roots in this.
- Each new copy of DNA that results during DNA replication is nearly identical to the original, save for a few alterations. This careful tending to the species’ distinctiveness, combined with the minor mutation that gives each individual creature its own distinct personality, is essential.
- DNA replication can go one of three ways: conservatively, semi-conservatively, or randomly.
- DNA replication with partial conservatism is the standard method.
What is DNA replication stress?
DNA replication stress is how a cell’s genome reacts when it is exposed to different stresses. Replication stress is caused by things that happen during DNA replication and can cause the replication fork to stop moving.
There are a lot of things that can cause replication stress, such as:
- Using the wrong ribonucleotides
- Unusual DNA structures
- Replication and transcription don’t always get along.
- Not enough important replication factors
- Common fragile sites
- Overexpression of oncogenes or constant activation of them
- Chromatin can’t be reached
ATM and ATR are proteins that help keep replication stress from happening. In particular, they are kinases that are brought in and turned on when DNA is damaged. If these regulatory proteins don’t keep the replication fork stable, it could fall apart. When this happens, the broken end of the DNA is fixed by putting the fork back together again.
Why is DNA called a Polynucleotide Molecule?
The DNA is called a polynucleotide because it is made up of nucleotides: deoxyadenylate (A), deoxyguanylate (G), deoxycytidylate (C), and deoxythymidylate (T). When these nucleotides are put together, they form long chains that are called polynucleotides. Based on how DNA is put together, it has two chains of polynucleotides.
What is Chargaff’s Rule?
A biochemist named Erwin Chargaff found that there were the same number of nitrogenous bases in the DNA. How much A is is equal to T, and how much C is is equal to G.
In other words, there should be an equal number of purine bases and pyrimidine bases in the DNA of every cell in every living thing.
Why dna replication is called semiconservative?
- Each strand of DNA is used as a model to make a new strand. This makes two new molecules of DNA, each with one new strand and one old strand. This is a semiconservative copy.
- Soon after their 1953 paper on the structure of DNA came out, Watson and Crick proposed the idea of semiconservative replication. In 1957, Matthew Meselson and Franklin Stahl did experiments that were cleverly designed to prove the idea.
- Meselson and Stahl grew E. coli cells for many generations in a medium where the only source of nitrogen (NH4CI) was the “heavy” isotope of nitrogen, 15N, instead of the more common “light” isotope, 14N.
- When the DNA from these cells was taken out, it was about 1% more dense than normal [14N]DNA. Even though this isn’t a big difference, a mixture of heavy [15N] DNA and light [14N] DNA can be separated by centrifuging until equilibrium is reached in a cesium chloride density gradient.
- The E. coli cells that were growing in the 15N medium were moved to a new medium with just the 14N isotope and allowed to grow there until the number of cells had just doubled.
- The DNA from these first-generation cells formed a single band in the CsCl gradient at a place that showed that the double-helical DNA molecules of the daughter cells were hybrids with one new 14N strand and one parent 15N strand.
- This result argued against an alternative theory called “conservative replication,” which said that one of the offspring DNA molecules would be made up of two newly made DNA strands and the other would be made up of the two parent strands. In the Meselson-Stahl experiment, hybrid DNA molecules would not be made in this way.
- In the next step of the experiment, more proof was found to back up the semiconservative replication hypothesis.
- In the 14N medium, cells were again given a chance to double in number.
- The DNA from this second cycle of replication was found to have lruo bands in the density gradient. One band had the same density as light DNA, and the other had the same density as the hybrid DNA seen after the first cell doubling.
Why DNA Synthesis Proceeds in a 5’-3’,Direction and Is Semi Discontinuous?
- A new strand of DNA is always made from 5′ to 3′, with the free 3, OH being the point where the DNA gets longer.
- Since the two strands of DNA don’t go in the same direction, the template strand is read from its 3′ end to its 5′ end.
- If synthesis always goes from 5′ to 3′, then how can both strands be made at the same time? If both strands were made at the same time as the replication fork moved, one strand would need to go through 3′-to-5′ synthesis.
- In the 1960s, Reiji Okazaki and his team solved this problem.
- Okazaki found that one of the new DNA strands is made up of short pieces. These pieces are now known as Okazaki fragments.
- This work led to the final conclusion that one strand is made continuously, while the other is made in pieces.
- The continuous strand, also called the leading strand, is the one where the 5′-to-3′ synthesis goes in the same direction as the movement of the replication fork.
- The discontinuous strand, also called the “lagging strand,” is the one in which 5′-3′ synthesis happens in the opposite direction of the way the fork moves.
- Depending on the type of cell, Okazaki fragments can be anywhere from a few hundred to a few thousand nucleotides long.
DNA Replication Mind Map
DNA replication refers to the copying of genetic information from one strand into another strand, which occurs during cell division. This process allows cells to pass copies of genes onto daughter cells. Replication takes place in each cell cycle. During S phase, DNA replicates itself.
DNA replication occurs during mitosis, when cells divide. During prophase, chromosomes condense and become visible under the microscope. Condensed chromosomes are separated into two daughter cells. The cell then enters prometaphase, telophase, and metaphase. At the end of metaphase, each chromosome has been assigned to one of the new daughter cells.
The enzyme which is used to unwind DNA is the DNA helicase enzyme, which belongs to the class Helicase and is important for all organisms.
It moves along the double-stranded DNA and separates the strands by breaking hydrogen bonds at the expense of energy from ATP.
DNA ligase is a specific type of enzyme, a ligase facilitates the joining of DNA strands together by catalyzing the formation of phosphodiester bonds.
A primer is a short nucleic acid sequence that provides a starting point for DNA synthesis. In living organisms, primers are short strands of RNA. A primer must be synthesized by an enzyme called primase, which is a type of RNA polymerase, before DNA replication can occur.
Replication is called semiconservative because at the time of replication, in each of the two copies of the DNA, one of the strands of DNA is old and conserved and one is newly formed.
DNA replication takes place during the Synthesis (S) phase of cell cycle. During S phase, an enzyme called helicase unwinds the DNA strand. The two single DNA strands can then be used as templates to form two identical double DNA strands.
DNA polymerase has the proofreading ability.
- Microbiology by Prescott, 11th Edition
- Hejna, J. A., & Moses, R. E. (2009). DNA Replication. Encyclopedia of Microbiology, 113–122. doi:10.1016/b978-012373944-5.00071-7
- Maga, G. (2013). DNA Replication. Brenner’s Encyclopedia of Genetics, 392–394. doi:10.1016/b978-0-12-374984-0.00431-9