- Discovery of DNA:
- Sequencing and Structure of DNA:
- Replication of DNA:
- Process of DNA Replication:
- Difference Between Eukaryotic and Prokaryotic DNA Replication:
- DNA Repair Mechanism:
- The hereditary material present in most organisms including human beings is DNA. It constitutes all the genetic information regarding the appearance and functioning of minute cells in an organism and is passed down from one generation to another.
- It consists of Purines and Pyrimidine, which are the basic structural units, responsible for creating the complex helix of the double-stranded DNA.
- The replication mechanism of DNA is carried out by a set of enzymes known as replication machinery and the procedure is almost identical for both eukaryotes and prokaryotes. Differences are discussed later in the article.
- In case any mistake occurs during this procedure of DNA replication, it might generate a mutation in the genome which could end up in development of a disease. Thus, a special process of proofreading and correction is carried out by DNA polymerase – an essential enzyme for the process.
The fundamental hereditary part present in most of the eukaryotes and prokaryotes is DNA, which is an abbreviation of deoxyribonucleic acid. We are well aware of the fact that functions and structure of a cell are controlled by the genetic material present inside the nucleus, usually located in the centre of the cell. This genetic material consists of genes that are constituted with the help of nucleotide base pairs present in the DNA.
A human cell consists of 46 chromosomes, half of which are inherited from the mother’s genome and the other half from the father’s genome. All these genes are responsible for eliciting the unique set of phenotype and genotype a child carries. Even though this adds up to a total of 3 billion nucleotide base pairs, however, only less than 25 % of these genes are functional in nature for the human body.
DNA can be extracted from any part of a human body, whether it is from saliva, hair, nails, etc. since it is located in the nucleus and the mitochondria of every cell in our body. In short, you can classify DNA as the scientific identity of an individual, which acts as a basis for many situations such as determining parental relationships or tracking genetically linked disease and is also now widely used in cracking criminal cases with the process known as of DNA fingerprinting.
Discovery of DNA:
The historical basis of DNA leads us back to the 1860s, when Friedrich Miescher first detected the presence of highly rich phosphate molecules in the nucleus of white blood cells (leukocytes) and named this material as nucleic acid, which was in lieu of its presence in the nucleus of a cell. However, several studies later, we were led to the term deoxyribonucleic acids (DNA). Initially, there were three hypotheses regarding the replication of a DNA. The conservative replication hypothesis suggested that the original double strands of DNA were used to create new double strands of DNA with no segments from the original ones. Whereas the dispersed hypothesis suggested that some segments of original double-stranded DNA were distributed among the new double-stranded DNA.
It was established in later years, when further experiments were performed by a number of scientists, that the process was ‘semiconservative replication’. To elaborate, semiconservative replication means that the double strands of DNA are straightened, split open and each of these strands acts as a masterpiece for the production of a complementary strand.
Sequencing and Structure of DNA:
The structure of DNA was first studied by Francis Crick and James Watson using X–Ray Crystallography technique. There are three main components that combine to form a double-stranded DNA molecule. A nitrogenous base that is further subdivided into two categories, purine and pyrimidine. Purines consist of adenine (A) and guanine (G), whereas pyrimidines consist of cytosine (C) and thymine (T), but in case of prokaryotes or RNA, uracil (U) replaces thymine (T) instead. These nitrogenous bases pair together to give us a double hydrogen bond between adenine (A) and thymine (T) whereas guanine (G) forms a triple hydrogen bond with cytosine (C) resulting in a helical structure of the double-stranded DNA. An interesting notable fact is that ten (10) base pairs are present in one helix or turn, of the double-stranded DNA.
A pentose sugar molecule to which the nitrogenous bases are attached at the first carbon (1’) and the phosphate molecule, the third main component of a DNA, is attached at the fifth carbon (5’). In the case of DNA, the second (2’) carbon is a hydrogen group (H) while in the case of RNA a hydroxyl group (OH) is attached at the position instead. This gives the DNA molecule its deoxyribose characteristic. The free hydroxyl group located at the 3’ carbon is responsible for formation of backbone of the ladder as phosphate groups attach to it. The two strands of the DNA appear to be in the opposite direction which indicates that one strand will be in 5’ to 3’ carbon whereas its complementary strand will be in the 3’ to 5’ carbon direction.
When a nitrogenous base is attached to a sugar molecule it is known as a nucleoside molecule. Additionally, when a nucleoside (nitrogenous base + phosphate molecule) is attached to a sugar molecule it is then known as a nucleotide molecule. In other words, attachment of a phosphate molecule to a nucleoside creates a nucleotide molecule. Now depending on the number of phosphate molecules attached, it either forms a nucleoside monophosphate (one phosphate molecule), a nucleoside diphosphate (two phosphate molecules), or a nucleoside triphosphate (three phosphate molecules).
(Mary Ann Clark, Jung Choi,Matthew Douglas, 2018)
Excessive presence of phosphate groups in the double-stranded DNA gives the molecule an overall negative charge. A very basic protein molecule consisting of ‘two sets of four histone molecules’ is known as histone octamer. DNA molecule wraps around these histone octamers to create a rather compressed structure, known as the nucleosomes, and appear as thread twisted around beads. These nucleosome fibres further coil up to form chromatin fibre. Moving forward, these highly compressed chromatin fibres form a condensed chromosome.
(Mary Ann Clark, Jung Choi,Matthew Douglas, 2018)
Replication of DNA:
Replication of DNA requires a specific set of enzymes to create a complementary strand against an original strand of DNA while undergoing the semi-conservative replication process.
Enzymes involved in the replication process of DNA are listed below with respect to their specific functions:
- Helicase is responsible for breaking the hydrogen bonds present between the nitrogenous bases and separating the double-stranded DNA helix.
- Single-strand binding proteins (SSBP) get attached to the single strands of the DNA to prevent them from rewinding and to hold the template strands in their place.
- Topoisomerase is responsible for preventing the supercoiling of DNA by getting attached before the replication fork.
- Primase creates an RNA primer that is complementary to the template DNA strand and helps to initiate the replication process.
- DNA Polymerase is the main enzyme that is carried on the replication of DNA by reading the template strand and then arranges the nucleotides to form a complementary strand.
- Ligase is the enzyme that ensures that the lagging strand is joined together to form one continuous complementary DNA strand.
Process of DNA Replication:
To give you a better idea of the process involved in the replication of DNA, we have subdivided it into three main parts. Each of these is discussed briefly below to give you an elaborate understanding of how and which enzymes are used at every step.
To start the process of replication the DNA undergoes some changes which determine the ‘origin of replication’ part, such as the presence of specific sequencing for prokaryotes or uncoiling in the nucleosome region which makes it possible for the replication machinery to get attached to it.
One of the first enzymes which get attached to the origin of the replication region is the helicase enzyme. You should already be aware by now that a helicase unwinds the double-stranded helix structure of DNA and breaks the hydrogen bonds among the nitrogenous base pairs to form a single-stranded DNA. So, to ensure that the bases do not rebind, the single-stranded binding proteins (SSBP) attaches itself to the single strands of DNA. This creates a Y-shaped structure at the origin of replication known as ‘fork or bubble of replication’ whereby additional enzymes play their roles in performing DNA replication process.
Complementary strands are formed during the elongation phase and are conditioned to grow in the direction of 5’ carbon to 3’ carbon only. To prevent the DNA from coiling again, Topoisomerase binds on the DNA ahead of the replication fork. This prevents the primary enzyme, DNA Polymerase from binding the template strands back over on themselves. Instead, now it is only able to add new nucleotides to the open template strand at the 3’ carbon position.
Therefore, the DNA Polymerase binds to the template DNA strands and creates a short polynucleotide RNA sequence which is complementary to the template DNA and is known as the primer. DNA polymerase recognizes the free 3’ carbon ends of these primers and starts elongating the complementary DNA strand by reading the template DNA strand and adding nucleotides accordingly to the 3’ end of the complementary strand of DNA.
Since the DNA polymerase adds nucleotides to the 3’ carbon end only, the process of elongation produces two types of strands as the template strands are antiparallel in nature. The first one is the leading strand, which grows in the direction of the replication fork, as the DNA polymerase keeps adding to the 3’ end of the complementary strand, in a continuous manner, while the template DNA uncoils.
The second one is the lagging strand. As the name suggests, this strand is formulated in a discontinuous manner as here the DNA polymerase moves in the ‘opposite direction from the replication fork’. This creates short fragments of complementary DNA strands known as the Okazaki fragments, from 5’ to 3’ direction. Each of them contains its own primer and is thus interrupted by the previously created Okazaki fragment.
Once the DNA polymerase reaches the part that has already been replicated, it stops. This creates one continuous leading strand of DNA, and another discontinuous lagging strand. The RNA primers are subsequently removed from the Okazaki fragments and leading strand. This creates unattached DNA fragments, called nicks, lacking portions of the sugar-phosphate backbone of the ladder. The freely floating DNA polymerase recognizes these free 3’ end carbons and fills these gaps with complementary DNA sequence. Finally, the ligase enzyme recognizes these nicks and joins the Okazaki fragments to form a continuous lagging and leading strand of DNA. Thus, replicating two daughter strands from a single parent strand of DNA.
Difference Between Eukaryotic and Prokaryotic DNA Replication:
Though, most of the enzymes and steps carried out during the replication of DNA for eukaryotes and prokaryotes are similar but there are some prominent differences that tell apart the DNA replication process occurring in these two. The are discussed below which will help you distinguish between the replication of DNA in a eukaryotic cell and a prokaryotic cell.
|Characteristics||Prokaryotic cell||Eukaryotic cell|
|Speed of replication||Fast replication with about 2000 base pairs per second||Slow replication with about 100 base pairs per second|
|Size of Okazaki fragments||Large fragments||Small fragments|
|Number of origins of replication||Single||Numerous|
|Types of DNA polymerase||Five (5)||Fourteen (14)|
|Presence of telomeres||Absent||Present|
|The enzyme that removes RNA primer removal||DNA polymerase – I||RNase H|
Note that there is a special, protective, cap-like structure at the end of linear DNA eukaryotic chromosomes known as Telomeres. These cap-like structures prevent the loss of genes and occur at the end or tip of chromosomes. They also prevent any unnecessary DNA repair mechanism from triggering which might alter the genetic composition of an organism by a mutation that can lead to serious genetic diseases.
Since the prokaryotic DNA appears in a circular form, the requirement of Telomeres does not exist anymore and so unlike eukaryotic chromosomes, which can be affected by the cytoplasmic enzymes and need proper protection, prokaryotic chromosomes do not entail such protection against degradation enzymes.
DNA Repair Mechanism:
Not everything is always fool proof to the dot. Sometimes, even the intricate functioning machinery of our body may make mistakes, and the impact may vary depending on the location and intensity of the derailing. Such are the cases observed during the process of DNA replication, where the DNA polymerase enzyme is responsible for production of daughter DNA molecules might end up creating a mistake. These mistakes are known as a ‘mutation’ in the genome of an organism. The mutation caused are mainly of two main types – spontaneous mutation which occurs due to the course of action occurring naturally in our body, and induced mutation which occurs due to the exterior environmental factors such as UV or X-ray exposure, chemicals, etc. The level of mutation might vary from being a small nitrogenous base mistake, known as a point mutation or even cause a chromosomal part being mutated to a full-fledge chromosomal deletion or addition, causing diseases like Turner syndrome.
To prevent such situations, there are special inbuilt mechanisms that include proofreading of the newly synthesized DNA molecules, making sure in the start that the process of DNA replication is successful. The proofreading of the new daughter DNA molecules is carried out by DNA polymerase. If an error is recognized, DNA Polymerase then initiates the process of DNA repairing.
There are two main types of DNA repair mechanisms. The first one is known as the mismatch repair mechanism. As the name suggests the error occurring in this situation is that the nitrogenous bases are mismatched and therefore no hydrogen bond exists between them. Once the DNA polymerase recognizes the situation is, first of all, excises the region containing the mismatched nitrogenous base pair. Then the DNA polymerase moves on to synthesizing a whole new strand containing the correct nitrogenous base pairing by adding the nucleotide to the 3’ free end carbon. This results in the rectification of mismatch error that occurred during the DNA replication mechanism.
The other type of DNA repair mechanism is known as the nucleotide excision repair mechanism. Although it is almost similar to the mismatch repair mechanism as it uses the excision technique. But the error actually occurs due to the impaired nucleotide found during the proofreading of the newly synthesized daughter DNA molecule. Therefore, the nucleotides are simply removed, and correct nucleotides are added. But this requires ligase to bind the sugar-phosphate backbone of the leader of the daughter DNA molecule, making the replication of DNA successful.
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