Concept of Directions, Enzymes and Replication machinery during DNA Replication in Prokaryotes

DNA Replication in Prokaryotes

DNA is the genetic material of both prokaryotic and eukaryotic organisms and it decides the characteristics of both these organisms. The DNA present in prokaryotes can vary from eukaryotes in size and shape because prokaryotic cells lack a prominent nucleus and have chromosomes that are in circular shape but the replication process of DNA is very similar to eukaryotes except for some points. 

Only a single point of origin is present in prokaryotes through which DNA replication takes place. In the case of eukaryotes, multiple points of origin are present. This means DNA replication starts from multiple parts of the DNA. 

The cell divides its DNA during the S-phase of the cell cycle. As the Meselson-Stahl experiment confirmed DNA has a semiconservative mode of replication so both of the strands of DNA remain conserved however they are present on two different copies of DNA. 

DNA is the basic unit of information because all the cellular processes are controlled by its DNA. So very small mutations in the DNA can cause big changes that’s why every single prokaryotic and eukaryotic cell has well-regulated and well-controlled mechanisms that resist the changes in DNA by making many checkpoints during DNA replication. These mechanisms show that how cells strictly focused on maintaining their original genetic materials.

The DNA replication machinery of prokaryotic cells

1. Polymerase enzyme
2. Primase enzyme
3. Helicase enzyme
4. Ligase enzyme
5. DNA gyrase
6. Beta clamp
7. Clamp loader
8. Single-stranded binding proteins (SSB)

• DNA polymerase is a family of enzymes that carry out all types of DNA replication processes. It is one of the most effective enzymes in the replication process because it not only forms the new strand of DNA but also can check whether the sequences are good or not. However, it can only extend the strand of DNA from an existing DNA strand. It can never begin the synthesis of a new strand.
• Primase enzyme is responsible for primer. Polymerase enzyme cannot perform its function without a primer. In DNA replication, the primer is the short nucleotide sequence of RNA that binds with the single strand of DNA and gives space to the polymerase enzyme to perform its function.
• DNA helicase has a seizer-like activity. It separates the double-stranded structure of DNA by breaking hydrogen bonds between the base pairs of DNA. The origin where helicase starts to separate the double-stranded DNA is called the origin of replication. Usually, the origins of replication are the region where adenine and thymine base pairs are present in healthy concentration because it's easy for helicase to break two hydrogen bonds rather than three hydrogen bonds. DNA polymerase can work only on a single strand of DNA, on a double-stranded structure DNA polymerase can’t show any activity.
• Ligase enzymes are the special ones. It has a glue-like ability because it attaches two different fragments of DNA (e.g. Okazaki fragment)  during the replication process.
• DNA gyrase makes sure that the double helical structure is not supercoil. It is present ahead of DNA helicase.
• Beta clamp and clamp loader help to hold the DNA polymerase in place during the replication process.
• SSB proteins ensure that the separated DNA strands do not coil with each other.

Directions during Replication.

The concept of directions is very important to understand the replication process of DNA because each double-stranded DNA molecule has two strands that are anti-parallel to each other. And DNA polymerase enzyme can only produce a new strand in 5’ to 3’ end in direction because it reads DNA from 3’ to 5’ end in direction. 

As we know DNA is the polymer of nucleotides and a nucleotide is made up of three units i.e. pentose sugar, phosphate groups, and nitrogenous bases. 

Pentose sugar is a five-carbon sugar molecule. On carbon number 1, the nitrogen base is attached while the phosphate group binds at carbon number 5 of the pentose sugar. Nitrogenous bases vary in the structure of DNA. Four nitrogenous bases are the basic informative units that are present in DNA.

1. Adenine
2. Guanine
3. Cytosine
4. Thymine

While pentose sugar and phosphate groups form the backbone of DNA and the direction is determined by this backbone. Phosphate forms diester bonds with pentose sugar. 

It forms a bond with a carbon number 5 and forms a second photo-ester bond with carbon number 3 of the pentose sugar and in this way forms a long chain of nucleotides (pentose sugar + phosphate groups + nitrogenous bases). 

At the edges of this chain, the phosphate linked with carbon number 5 of pentose sugar and carbon number 3 of pentose sugar is always free. These free sites are counted and give the direction of DNA in the double-stranded helical structure.

 

Now, why do strands of DNA run antiparallel to each other?

The answer is to link the nitrogenous bases to form a double-stranded structure of DNA. As nitrogenous bases are attached at carbon number 1 of a pentose sugar, to form a linkage between these bases one of the two strands of DNA turns towards the other strand and runs antiparallel.

 

DNA replication in prokaryotes.

• The super-coiled double-stranded structure of DNA is separated by the Helicase enzyme. This enzyme breaks the hydrogen bonds present in the structure of DNA.
• ATP is used by helicase to separate the strands of DNA.
• DNA gyrase travels ahead of the helicase and alleviates these supercoils.
• Some single-stranded binding proteins (SSB proteins) came into action and ensured that both separated strands of DNA were not going to attach to each other.
• Then RNA primers (10 to 12 nucleotides) are synthesized by primase enzyme which is 5’ to 3’ in direction and needed to form a new strand of DNA.
• Once a primer pairs with the strand of DNA to be replicated, DNA polymerase 3 starts to synthesize a new strand of DNA.
• DNA replication is an energy-consuming process and this energy is taken by breaking the bonds between nucleoside-triphosphates (like adenosine-triphosphate, guanosine-triphosphate, cytosine-triphosphate, etc). They all are present in the nucleosome.
• As Adenosine tri-phosphate is broken into adenosine mono-phosphate, the remaining two phosphate groups have high energy and this energy is used by DNA replication machinery.
• DNA polymerase 3 breaks ATP (adenosine triphosphate) into adenosine mono-phosphate and this adenosine mono-phosphate and then it becomes the part of new synthesizing DNA. The same thing happens in the case of all other nitrogen bases (i.e. guanine, cytosine, thymine).
• DNA template reading during its replication process by DNA polymerase 3 enzyme is always 3’ end to 5’ end in direction and the formation of a new strand which we call the daughter strand is 5’end to 3’ end in direction.
• The DNA replication process is always bi-directional because prokaryotes have double-stranded circular DNA which has only one origin of replication.
• One strand of DNA has a 3’ to 5’ end in direction while the other strand of DNA is a 5’ to 3’ end. It means that both strands always move opposite in direction or anti-parallel to each other.
• As we discussed earlier the DNA polymerase enzyme reads the template DNA which starts from the 3’ end and continues towards the 5’ end, so the primer attached with 3’ to 5’ direction had a smooth way of replication because the new strand is smoothly formed which is 5’ to 3’ in direction. This is also called as leading strand.
• The primer attached with a 5’ to 3’ end, forms fragments of replicated DNA which we call Okazaki fragments.
• These fragments are formed because DNA polymerase is unable to replicate DNA at the other strand (i.e. lagging strand) for two reasons.

1. The first reason is that DNA polymerase never reads the strand of DNA from the 5’ to 3’ end. It always moves from a 3’ to 5’ direction and forms a new strand that is antiparallel to its template strand (i.e. 5’ to 3’ end).
2. The second reason is that the un-winding or separation of DNA strands takes time by the helicase enzyme and its speed to separate DNA strands is well regulated by replication machinery.

• This type of continuous and discontinuous mode of replication of DNA is called the Semi-discontinuous model of DNA synthesis.
• It means that one strand of DNA has only a single primer while the other strand has multiple primers on it.
• The strand of DNA that has only one primer acts as the leading strand while the strand that has multiple primers acts as the lagging strand.
• Once the DNA polymerase 3 has done its work there is a need to remove primers (i.e. RNA strands). These RNA strands are removed and replaced by DNA polymerase 1. 
• In this way, two exact copies of double-stranded structures of DNA are formed from its single parent DNA.
• DNA ligase is needed to join these okazaki fragments and form a completely new single strand of DNA.

 

Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks meet each other on the opposite end of the parental chromosome.

E coli regulate this process through the use of termination sequences which, when bound by the Tus protein, enable only one direction of replication fork to pass through

As a result, the replication forks are constrained to always meet within the termination region of the chromosome.