Deoxyribonucleic acid known as DNA is a structural molecule which carries the genetic information of all organisms. DNA has a helical structure in which two strands of DNA twist around each other. The stability of DNA molecule is assessed by the length of the strand and the melting temperature. There are various enzymes which participate in the replication of the DNA molecule and these enzymes collectively form a complex machine known as the replisome.
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Image: “DNA Microscopic Cell Gene Helix Science Medical” by typographyimages. License: Public Domain


DNA Structure

DNA, also known as deoxyribonucleic acid is a molecule which carries the genetic information for the growth, development, and reproduction of all organisms including some of the viruses. DNA molecules are arranged in the form of a double helix in which two DNA strands are coiled around each other. DNA strands are made up of nucleotides and hence are called polynucleotides. Each nucleotide is made up of four nitrogen-containing nucleobases which can be either cytosine (C), thymine (T), guanine (G) or adenine (A), a deoxyribose sugar and a phosphate group.

Covalent bonds join the sugar of one nucleotide to the phosphate group of the other nucleotide resulting in a sugar-phosphate backbone. Adenine is paired with thymine and cytosine is paired with guanine by the hydrogen bond. DNA carries biological information. Each strand of DNA carries the same information and this information is replicated as such when the two strands of DNA separate. DNA backbone is resistant to cleavage. About 98% of DNA is non-coding and does not contain information for protein sequences. The two strands of DNA run in opposite direction to one another. RNA strands are made from the DNA strands through a process called transcription. In all eukaryotic organisms like animals and plants, DNA is stored in the nucleus and is organized into structures called chromosomes. During replication, the chromosomes are duplicated providing each cell its own set of chromosomes. DNA is also stored in mitochondria which are known as the powerhouse of the cell. In prokaryotic organisms like bacteria, DNA is stored in the cytoplasm.

Nucleobase classification

Nucleobases are classified into two types:

  • Purines: Purines are adenine and guanine
  • Pyrimidines: these are cytosine and thymine

Grooves

The two DNA strands are not symmetrically arranged as they twist around each other and form a helical structure. This arrangement creates grooves in the structure and as the twisting is not symmetrical the grooves are also of different width sizes. There are major grooves were different transcription factors can bind to the DNA strand as this site is more accessible. The other grooves are called minor grooves.

Base pairing

In complementary base pairing, purines form hydrogen bonds with pyrimidines in which adenine binds to thymine using 2 hydrogen bonds and cytosine binds to guanine with the help of 3 hydrogen bonds. This arrangement of binding across the double helix is called Watson – Crick base pairing. In Hoogsteen base pairing, two hydrogen bonds form between guanine and cytosine. DNA containing a large amount of GC content is more stable than the one containing a small amount of GC content. Melting is a process in which the dsDNA is converted into ssDNA. Melting occurs at high temperature, high ph, and low salt concentrations. The strength of a DNA molecule can be assessed by the melting temperature and length of the molecule.

Sense and antisense

If the DNA sequence is same as that of messenger RNA copy then the sequence is called “sense” and the sequence on the other opposite strand is called antisense. Both sense and antisense sequence can occur on the same DNA. Antisense RNA is produced in both eukaryotes and prokaryotes but their function is not clear. One proposal that has been given is that they are involved in gene expression.

Supercoiling

When the DNA is twisted along its own axis then this process is known as supercoiling. If the DNA is twisted along the axis then this process is called positive supercoiling and in this process, the DNA molecule becomes tightly twisted. If it is twisted in opposite direction then this process is called negative supercoiling which results in a relaxed state. Enzymes called topoisomerases introduce negative supercoiling in the DNA molecule. These enzymes are also required for relieving the stresses which are introduced during replication and transcription processes.

DNA Replication

DNA replication is a biological process in which two identical strands are produced from one DNA molecule. Each of the separated strand acts as a template for the new strand and this process is known as semiconservative replication. Replication begins at specific points known as the origin of replication in the genome. This unwinding result in the production of replication forks in both directions. DNA replication is associated with a family of enzymes known as “DNA Polymerases”. These enzymes don’t initiate the synthesis of new strands rather they can only extend an existing strand. To begin the synthesis of new strand a “primer” is made which is a short segment of RNA. DNA polymerase extends the 3’ end of the existing nucleotide chain via the phosphodiester bonds. Some of the DNA polymerases have the proofreading ability as well which means they are able to read the mismatched base pairs and remove them.

Replication process

The replication process occurs in three steps which are:

  • Initiation
  • Elongation
  • Termination

Initiation

Specific proteins known as initiator proteins identify particular points in DNA known as “origins”. AT-rich regions are used by initiator proteins as they contain two hydrogen bonds which are easier to break than GC which has three hydrogen bonds. Once this region is recognized these proteins recruit other proteins to make a pre-replication complex which then starts unwinding the DNA helix.

Elongation

The new DNA strands are always synthesized in 5’ – 3’ direction while DNA template is always read in 3’ – 5’ direction. DNA replication requires a free hydroxyl group for the synthesis of a new strand. Once the unfolding of the two strands starts, primase starts adding RNA primers to the template strand. One RNA primer is added to the leading strand while many RNA primers are added to the lagging strand. The primer continuously extends the leading strand while the lagging strand is discontinued forming the Okazaki fragments. RNA fragments are removed by RNase and another polymerase enters to fill in the gaps. A single nick is created on the leading strand while several nicks are created on the lagging strand. Then another enzyme known as the DNA ligase works to fill in these nicks thus creating a new strand.

Replisome

Replisome is a complex molecular machine that is required for the replication of the DNA molecule. The replisome is responsible for the unwinding of the double helical structure of the DNA molecule which results in the formation of two single strands. For each of these single strands, a new complementary strand is formed which results in the formation of two new DNA molecules which are exact copies of the original DNA molecule from which the new strands were created. Following are the main enzymes that are required for the formation of replisome:

  • DNA Helicase: It destabilizes the DNA molecule. It is responsible for separating the DNA molecule at the replication fork behind the topoisomerases.
  • DNA polymerase: It adds nucleotides to DNA in 5’ to 3’ direction. It is also responsible for proofreading and error correction.
  • DNA clamp: This is a protein which prevents DNA elongating polymerases from being separated from the parent DNA.
  • Single-strand binding proteins: It maintains strand separation.
  • Topoisomerases: Relieves the DNA strand from its supercoiled nature.
  • DNA Gyrase: Relieves strain of unwinding.
  • DNA Ligase: Helps in the re-annealing of two strands.
  • Primase: Provides site for the synthesis of the new strand.
  • Telomerase: Adds nucleotides thus lengthen the DNA molecule.

Termination

In order for termination to occur the replication fork must be stopped from progressing. This occurs via two steps:

  • A termination site sequence on the DNA molecule.
  • A protein like Ter protein which binds physically to this site for the termination to occur.

Monohybrid crosses

A monohybrid cross is a breeding experiment between two organisms which differ only in a single given trait. For the trait that is being studied, parents are homozygous for that particularly given trait but differ in alleles for that given trait. It is used to study the dominance relationship between two alleles. In this cross, one parent is homozygous for one allele and the other parent is homozygous for the other allele. The offspring in the first generation are heterozygous for a trait and all express the dominant trait.

Dihybrid crosses

Dihybrid crosses refer to the breeding between two different strains which differ in two observed traits. The two different traits are involved and each trait has two different alleles. A punnet square of dimensions 4 x 4 clearly demonstrates a Dihybrid cross. When breeding occurs the first generation produces four identical offsprings and when the members of the first generation are crossed over it produces the second generation which shows the phenotypic appearance of ratio 9:3:3:1 which is interpreted as follows:

  • 9 show the offsprings with both dominant traits.
  • First 3 shows offsprings with the first dominant and second recessive trait.
  • Second 3 shows the offsprings with the first recessive and second dominant trait.
  • 1 shows the offsprings with both the recessive traits.
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