Biotechnolgy: DNA Replicaton | Lecture notes Biotechnology

The process of DNA replication occurs before cell division or mitosis and also meiosis. This process is

in the S (Synthesis) cycle. The first stage before the cell performs mitosis itself is DNA replication

first. In this replication process, in stages:

1. Initiation

The double-stranded DNA is attached to a protein that opens the double-stranded DNA. This is

necessary so that the helicase enzyme, which cuts hydrogen bonds on nucleotide bases, can

easily enter. The opening of double-stranded DNA into a single strand by the protein is

specifically located on the original DNA template. This template is located at the A-T nucleotide

base bond which has weak energy because it only has 2 hydrogen bonds compared to the

hydrogen bond at G-C which has 3 hydrogen bonds (so this bond has considerable energy). But

please note that the template of DNA can not have one original DNA. In fact, in DNA there is an

original DNA that depends on the amount of existence of the original DNA there is. Back to the

discussion after double-stranded DNA becomes single-stranded, please note that single-

stranded DNA tries to be able to become double-stranded again. To prevent this, the single-

stranded DNA is bound by the SSBP enzyme (--Single Strand Binding Protein-- meaning this

protein refers to an enzyme that binds to single-stranded DNA). In addition, the purpose of this

enzyme is to prevent nuclease enzymes (enzymes that can break the phosphodiester bond of

DNA causing it to become nucleotide again) from binding to single-stranded DNA that has been

opened double stranded by the initiation protein earlier. With the opening of the double strand

of DNA, it will distinguish the single strand of DNA at the top in the 3'-5' direction and at the

bottom 5'-3'.

2. Elongation

After the double-stranded DNA becomes single-stranded and each single-stranded DNA is

bound by the SSBP enzyme, the helicase enzyme enters and does its job to break the hydrogen

bonds on the nucleotide bases. However, it is different with primase and DNA polymerase

enzymes. Both enzymes work to form new polynucleotide chains with existing DNA templates

from both single-stranded DNA. The role of the primase enzyme is to initiate the initial

placement for DNA Polymerase to elongate the nucleotide. As for the DNA Polymerase enzyme,

it connects new nucleotide chains after being initiated by the primase enzyme. Referring to the

primase enzyme first as an initiator, the initiation carried out is by first reading the single-

stranded DNA chain in the 3'-5' direction, then after recognizing the right base pair, the

appropriate nucleotide base is paired with the template DNA nucleotide base. Please note that

the primer nucleotides are attached to the DNA template of each single strand in the form of

RNA by RNA Polymerase with the direction of installation is 5'-3'. Back to after the RNA

Polymerase enzyme initiates a primer on the DNA of each single strand in the form of RNA

nucleotides, then the elongation of nucleotide bases in the form of DNA chains is carried out

through the DNA Polymerase enzyme by continuing the RNA primase that has been initiated

and the same elongation direction as RNA Polymerase to hold the primer, namely 5'-3'. Please

also note, that the type of enzyme to perform this extension is a type 2 DNA Polymerase, where

this enzyme only has a role to extend new nucleotides.

If you imagine when at the stage of opening the double strand into a single strand, the strand

that has been opened is divided into 2 single strands with the upper and lower positions of each

double strand that has been opened. At the top of the single stranded DNA is called the Leading

Strand, while at the bottom of the other single stranded DNA is called the Leaging Strand. Back

to after DNA Polymerase elongates the new DNA strand based on the existing DNA template, at

the Leaging Strand when DNA Polymerase type 2 elongates, it is different from the nucleotide

elongation at the Leading Strand which continues to elongate as long as the helicase enzyme

breaks the hydrogen bonds in the DNA nucleotide base. This is because in the nucleotide

elongation of the Leading Strand, there are nucleotides that are shaped like fragments of

primary DNA and RNA known as Okazaki Fragments. The reason for the formation of Okazaki

Fragments on the Leaging Strand by imagining these strands towards 5'-3', is because when the

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The process of DNA replication occurs before cell division or mitosis and also meiosis. This process is in the S (Synthesis) cycle. The first stage before the cell performs mitosis itself is DNA replication first. In this replication process, in stages:

Initiation The double-stranded DNA is attached to a protein that opens the double-stranded DNA. This is necessary so that the helicase enzyme, which cuts hydrogen bonds on nucleotide bases, can easily enter. The opening of double-stranded DNA into a single strand by the protein is specifically located on the original DNA template. This template is located at the A-T nucleotide base bond which has weak energy because it only has 2 hydrogen bonds compared to the hydrogen bond at G-C which has 3 hydrogen bonds (so this bond has considerable energy). But please note that the template of DNA can not have one original DNA. In fact, in DNA there is an original DNA that depends on the amount of existence of the original DNA there is. Back to the discussion after double-stranded DNA becomes single-stranded, please note that single- stranded DNA tries to be able to become double-stranded again. To prevent this, the single- stranded DNA is bound by the SSBP enzyme (--Single Strand Binding Protein-- meaning this protein refers to an enzyme that binds to single-stranded DNA). In addition, the purpose of this enzyme is to prevent nuclease enzymes (enzymes that can break the phosphodiester bond of DNA causing it to become nucleotide again) from binding to single-stranded DNA that has been opened double stranded by the initiation protein earlier. With the opening of the double strand of DNA, it will distinguish the single strand of DNA at the top in the 3'-5' direction and at the bottom 5'-3'.
Elongation After the double-stranded DNA becomes single-stranded and each single-stranded DNA is bound by the SSBP enzyme, the helicase enzyme enters and does its job to break the hydrogen bonds on the nucleotide bases. However, it is different with primase and DNA polymerase enzymes. Both enzymes work to form new polynucleotide chains with existing DNA templates from both single-stranded DNA. The role of the primase enzyme is to initiate the initial placement for DNA Polymerase to elongate the nucleotide. As for the DNA Polymerase enzyme, it connects new nucleotide chains after being initiated by the primase enzyme. Referring to the primase enzyme first as an initiator, the initiation carried out is by first reading the single- stranded DNA chain in the 3'-5' direction, then after recognizing the right base pair, the appropriate nucleotide base is paired with the template DNA nucleotide base. Please note that the primer nucleotides are attached to the DNA template of each single strand in the form of RNA by RNA Polymerase with the direction of installation is 5'-3'. Back to after the RNA Polymerase enzyme initiates a primer on the DNA of each single strand in the form of RNA nucleotides, then the elongation of nucleotide bases in the form of DNA chains is carried out through the DNA Polymerase enzyme by continuing the RNA primase that has been initiated and the same elongation direction as RNA Polymerase to hold the primer, namely 5'-3'. Please also note, that the type of enzyme to perform this extension is a type 2 DNA Polymerase, where this enzyme only has a role to extend new nucleotides. If you imagine when at the stage of opening the double strand into a single strand, the strand that has been opened is divided into 2 single strands with the upper and lower positions of each double strand that has been opened. At the top of the single stranded DNA is called the Leading Strand, while at the bottom of the other single stranded DNA is called the Leaging Strand. Back to after DNA Polymerase elongates the new DNA strand based on the existing DNA template, at the Leaging Strand when DNA Polymerase type 2 elongates, it is different from the nucleotide elongation at the Leading Strand which continues to elongate as long as the helicase enzyme breaks the hydrogen bonds in the DNA nucleotide base. This is because in the nucleotide elongation of the Leading Strand, there are nucleotides that are shaped like fragments of primary DNA and RNA known as Okazaki Fragments. The reason for the formation of Okazaki Fragments on the Leaging Strand by imagining these strands towards 5'-3', is because when the

DNA Polymerase performs reverse nucleotide elongation with the helicase enzyme cutting the template DNA which causes there to be empty nucleotide bases as in the picture below. As long as DNA Polymerase performs nucleotide elongation based on the rules of the road (where as it is known that the synthesis path of DNA Polymerase is 5'-3') as long as the helicase enzyme cuts hydrogen bonds on nucleotide base bonds, there are conditions where the helicase enzyme will not cut in the twisted state of the DNA strand or referred to as the positive supercoil state (Supercoil stable) as in the figure below. With the positive twists or supercoils that are useful for folding DNA to fit the cell nucleus (because it is known that the DNA chain is very long. Therefore, this supercoil is needed), then in the replication process it is very necessary to have a gyrase enzyme or known as a topoisomerase enzyme. The mechanism of action of this enzyme is that the first hand of the enzyme known as nuclease acts as a DNA chain breaker, breaking the entangled DNA chain. At this stage, logically by breaking the coiled rope, the coiled form of the rope can be lost. Then, the other arm of the enzyme is known as ligase. So the other hand of the topoisomerase enzyme acts to glue the twists that are broken by the other hand of topoisomerase, the nuclease. So that after the DNA strand becomes a negative supercoil, the helicase enzyme can work again.

It can be seen that there are repeated and discontinuous primers and nucleotide chains. To overcome this, the primers, which are RNA, are replaced with DNA and also add separate chains and nucleotide base pairs to the new DNA that has been made by DNA Polymerase type 2. After the DNA Polymerase type 2 enzyme attaches a nucleotide chain to another new nucleotide DNA chain, there is an enzyme involved, namely the ligase enzyme (adhesive enzyme) to glue the two nucleotide chains together. However, there is one very crucial case in the process of replacing the Primary RNA at the end of the new strand DNA chain. When DNA Polymerase type 1 terminates the RNA Primer at the very end of the Lagging Strand, there is an overhang (void) because keep in mind that the carbon 5 sequence of the DNA Polymerase part of the Lagging Strand is a carbon atom that binds a phosphate group. So DNA Polymerase type 1 cannot connect the new strand (because for a nucleotide to bind to another nucleotide, a hydroxyl group is needed). (see the picture above in the 5'-3' direction at the top of the double strand. At the end there is an overhang or vacant pair from the top DNA strand to the bottom). With this problem, an enzyme appeared that could solve the problem. The enzyme involved is Telomerase, where the work of the enzyme is to lengthen the parent DNA strands that are overhang by Telomerase with the characteristics of the repeating base sequence (the base sequence in eukaryotic cells, especially humans is TTAGGG) called telomeres and the installation of nucleotide bases from telomeres is in the form of RNA chains. Why RNA? As is known, that the DNA of the new strand that has an overhang is a phosphate group, and to form a nucleotide chain there is a phosphodiester bond that requires an OH group (because of the name DNA which means Deoxyribonucleotide). So by adding RNA to the DNA strands is to attach phosphodiester bonds that require OH groups. After all the RNA chains are attached to the parent DNA chain, the RNA chains are replaced by DNA Polymerase type 2 to become all DNA strands. In addition to the role of telomeres extending short strands of DNA, it can also maintain the integrity of the genome such as from the genome does not stick with other genomes.

Biotechnolgy: DNA Replicaton, Lecture notes of Biotechnology

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