Prokaryotic DNA Replication Overview

Hello everyone, I am Dr. Trupti. Welcome to my YouTube channel Enjoy Biochemistry. In the video lecture series on molecular biology, today let's learn about replication in prokaryotes. Flow of genetic information involves three processes that explain how the genetic information contained in DNA is transmitted from one generation to next and utilized by the cell itself. So the first process is replication. In this process of replication, the parental DNA is copied to form daughter DNA molecule with identical nucleotide sequence. So the first process of flow of genetic information is replication. The second is transcription in which the genetic information contained in the DNA is transferred into RNA. And the third is translation whereby the genetic message encoded in RNA is translated on ribosomes into a polypeptide with a specific amino acid sequence. So by these three processes a gene which is the fundamental unit of genetic information it is used to produce a unique functional molecule and this functional molecule is usually protein or RNA. DNA replication is essential for the transmission of genomes and the genes they contain from one generation to other generation. It is required for continuation of species and occurs during cell division. Replication is very well studied in prokaryotes like E. coli. So what is replication? It is the process of synthesis of an identical duplicate copy of DNA from an existing DNA molecule. And DNA is replicated only once during the life cycle of a cell. So this is a duplex DNA strand from which two identical DNA stands are formed and this process is called as replication and the newly synthesized DNA stand there is one stand is newly synthesized and one stand is a parental stand and this replication is semi-conservative. Why this is called semi-conservative? It refers to the duplication of double standard DNA molecule in a such a way that the new DNA molecule has one original parental stand and one newly synthesized stand. This New double stranded DNA is identical to the original parental duplex DNA molecule. And this semi-conservative replication, it was proposed by Watson and Oric after they proposed the double helix model of DNA structure. Now let's see some salient features of replication. Each of the two DNA strand is used as a template to synthesize a new complementary strand to it. And new DNA is synthesized in 5'to 3'direction and for this the template strand is read from 3'to 5'end. So the synthesis of DNA occurs from 5'to 3'direction on both the template strands. Replication begins at origin and it is the specific site having 245 base pairs and it is called as ORIC. And at this particular site, when DNA untwist to expose the two single template strands for DNA replication, a Y-shaped structure is formed. So because of separation of two DNA, Y-shaped structure is formed and it is called as replication fork. And this replication fork, it moves in the direction of untwisting the DNA. So replication fork is moving. in both the direction and that's why the replication is bidirectional. So when DNA untwist, there is formation of two replication fork and these two Y shaped structure join together at their tops to form replication bubble. So this is called as replication bubble and it is nothing but the locally denatured segment of DNA. into one of the replication fork and this is the direction of movement of replication fork. Now let's understand what does it mean by leading strand and lagging strand. So on this strand, this upper strand, the DNA synthesis or DNA replication occurs from 5 prime to 3 prime direction in continuous manner. So this is called as leading strand. On the leading strand, replication occurs continuously in continuous manner. from 5 prime to 3 prime direction and also in the direction of movement of replication fork. So this is leading strand. Now what is lagging strand? The other one is lagging strand. DNA does not, synthesis does not occur here continuously. It occurs in short stretches. Short stretches of DNA are synthesized. So here the replication is not continuous. it is discontinuous but from 5'to 3'direction and this strand is called as lagging strand. And these short stretches of newly synthesized DNA they are called as Okazaki fragments. So here on the lagging strand replication is discontinuous and away from the direction of moment of replication 4. Though it is from 5'to 3'but it is away or opposite in the direction of replication fork while in case of leading strand replication is continuous and in the same direction of movement of replication fork. Though on the leading strand replication is continuous and on the lagging strand replication is discontinuous but it occurs simultaneously. For replication various factors are required and one of the factor is parental DNA as a template. Second factor required for replication is nucleotides, ribonucleotides for RNA primer synthesis. So these are various ribonucleotides having bases AUGC. Deoxyribonucleotides are required for complementary DNA strand synthesis. These are various deoxyribonucleotides having bases ATGC and various enzymes and accessory proteins are required for replication. So let's just enumerate various enzymes and proteins required for replication. We will see functions in later slides. So the various proteins and enzymes required are DNA A, helicases which is also called as DNA B and DNA C. These are required for DNA replication. Topoisomerases, these enzymes topoisomerase 1 and topoisomerase 2 are also required. Single standard binding protein are required. Primase. which is also called as DNAG it is required for RNA primer synthesis then DNA in DNA replication DNA polymerase 1 and 3 play very important role DNA ligase so these are various enzymes and proteins which are required for DNA replication DNA replication enzymes are called as DNA polymerase and this DNA polymerase catalyze replication but it requires a small piece of RNA it is called as primer. So RNA primer initiates DNA synthesis. So this is the primer which is 10 to 16 nucleotides long and it has 3 3 prime hydroxyl group and the DNA polymerase can add nucleotides to this 3 3 prime hydroxyl group of this RNA primer to replicate the DNA because DNA polymerase cannot add nucleotide without a primer. Now how this primer is synthesized? This RNA primer it is synthesized by enzyme called as DNA primase. This is also called as DNAG. It is modified RNA polymerase and it forms RNA primer. And on this RNA primer then DNA polymerase 3 can add new nucleotides on this RNA primer. So The new deoxyribonucleotide triphosphates, it is linked to free 3'hydroxyl end of the ribose and the nucleotides are joined to one another by 3'5'phosphodiester bond. There is release of pyrophosphate. So the energy required for this process of adding nucleotides, it is provided by this release of pyrophosphate and this is how DNA polymerase can add or it can catalyze the polymerization of various nucleotide precursors into DNA chain and the direction of synthesis of new DNA chain is from 5 prime to 3 prime because DNA polymerase can add new nucleotide at 3 prime end of the previous ribose sugar or deoxyribose sugar and that's why DNA synthesis occurs from 5 prime to 3 prime direction. Now at each step of lengthening of new DNA chain, DNA polymerase finds the correct precursor that is deoxyribonucleotide triphosphate that can form a complementary base pair with the nucleotide on the template strand of DNA. So RNA primers initiates DNA synthesis and without RNA primer, DNA polymerase cannot initiate or cannot replicate the DNA. There are 5 DNA polymerases known in E.coli and out of these 5, DNA polymerases 1, 2 and 3 they have role in DNA replication while type 4 and 5 they have role in DNA repair mechanisms. So DNA polymerase 1, 2 and 3 all have 3'to 5'exonuclease or proofreading activity while only DNA polymerase 1 it has 5'to 3'exonuclease activity. The polymerization rate that is the number of nucleotides added per second during replication is highest for DNA polymerase 3 that is 250 to 1000 nucleotides per second are added. Processivity it refers to number of nucleotides added by the enzyme before it dissociates from the DNA template and that processivity is also means highest for DNA polymerase 3 leading to the fastest chain elongation and this DNA polymerase 3. It is a multimeric enzyme with more than 10 subunits and two of its subunits are called as beta subunits and they act like a sliding clamp enhancing its stability and processivity. So the main role of DNA polymerase 1 it is the gap filling, main role of DNA polymerase 2 is repair, DNA repair and main role of DNA polymerase 3 it is the DNA replication. DNA replication is accurate because of DNA proofreading mechanism. And DNA polymerase 3 and DNA polymerase 1, they both have 3'to 5'exonuclease activity. It means that they can remove nucleotides from 3'end of DNA chain and it is useful for proofreading i.e. error correction. If an incorrect base is inserted by DNA polymerase, which can be 1 base in million, can be incorrect. So this error is recognized immediately by the enzyme and the enzyme 3'to 5'exonuclease activity excises the eraneous nucleotide from the new strand and then the DNA polymerase resume forward movement and inserts the correct nucleotide. In addition to this DNA polymerase 1 also has 5'to 3'exonuclease activity and it can remove nucleotides from the 5'end of DNA. So far we have studied various salient features of replication. So replication is semi-conservative, it is simultaneous and it is bidirectional. Replication is accurate because of proofreading mechanism due to 3'to 5'exonuclease activity of DNA polymerase 3 and 1. There is a formation of replication fork and replication begins at the origin and there is also formation of replication bubble in the process of replication and various enzymes and proteins are required for the replication along with various nucleotides, ribonucleotides and deoxyribonucleotides and this process of replication is catalyzed by DNA polymerases 3 and 1 but This DNA polymerase require RNA primer which is synthesized by enzyme primase and without which DNA cannot polymerize or cannot replicate the DNA. So these are the various salient features. Now let's understand the process of replication. Replication occurs in three stages and the first stage is initiation of replication. Second stage is elongation of replication. And third is termination of replication. Let's begin with the initiation of replication. And the initiation of replication is directed by a DNA sequence. It is called as replicator. And this replicator includes origin of replication where this replication begins. This is the area of origin of replication called as Oric. And it has two 45 specific base pairs. And this is the specific region where double. DNA double helix denatures into single strands within which replication begins. So this is the origin of replication. This ORIC where initiation begins it is 245 base pair long and it has two types of repeated conserved sequences. So there are three repeats of 13 base pair sequences reaching A and T bases and five repeats of nine base pair sequences. So there are two types of sequences 13 base pair repeats and 9 base pair repeats and one important protein that is DNA A protein along with ATP molecule it binds to this 9BP repeats and by binding to this 9BP repeats after that it recognizes and denatures this 13BP region which is reaching A and T bases so that's how this DNA protein help in the initiation of replication. The recognition and denaturation of 13 base pair region by DNA protein causes separation of DNA strand and there is formation of replication bubble and replication fork. DNA is an initiator protein which identifies Oric and causes separation of two strands by denaturation which allows binding of several other proteins like helicases, primase and polymerases. So helicase B which is also called as DNA B it is involved in separation of DNA strand and this helicases are recruited and loaded onto DNA strand by other protein which is called as DNA C. It is also called as DNA helicase loader protein. And this helicases untwist the DNA by breaking hydrogen bonds between the bases and the energy for untwisting comes from hydrolysis of ATP. So helicase requires ATP. This helicase and DNA-C both are hexameric string like molecules and each strand require one helicase molecule. So both strands require two helicase. DNA-C will help this helicase to load on this DNA strand. So this helicase molecule it will separate two strands of DNA. Now single standard binding protein. They bind. to the separated strand and prevent their re-naturation. So the separated strands are now they are stabilized for replication. So single standard binding protein they prevent re-annelling of the separated strand. Now the another enzyme topoisomerase. So this topoisomerase there are two types. Type 1 it reversibly cut one strand of DNA It has both nucleus and ligase activity and it does not require ATP. And the other type is DNA gyrase which make transient breaks in both the strand and it requires ATP. What is the function of this topoisomerase? So this topoisomerase relieves the supercoiling developed in DNA due to separation of strands. So it relieves supercoiling by creating NIC in the DNA strand. and by its nucleus and ligase activity then it can repair that DNA. So this process of initiation starts from the DNA A protein which identifies Oric then comes the role of DNA B and DNA C. DNA C will help this DNA B to get loaded on the DNA strand then this helicase will separate two DNA strands. The single-stranded binding protein will bind to the separated DNA strand and prevent its re-annulling. The enzyme topoisomerase binds to the DNA and relieves supercoiling which is developed in the DNA due to separation of strands. So after initiation of this replication, the elongation begins. This elongation is synthesis of a new strand of DNA using two separated strands as the template. New bases are added according to the principle of complementary base pairing depending on the sequence of bases in the template strand. So the nucleotide A in template strand is paired with T in the new strand. Similarly G is paired with C. So the replication occurs differently in two strands which are called as leading strand and lagging strand. So on the leading strand replication is continuous while on the lagging strand the replication is discontinuous. So leading strand runs from 3'to 5'direction and replication occurs in 5'to 3'direction. So for process of this elongation we require the enzyme DNA primase which is called as DNAG and it is required for synthesis of RNA primer because RNA primer initiates DNA synthesis. DNA polymerase cannot start this process of replication it requires RNA prime. So this is a DNA primase DNA G and it is required for synthesis of RNA primer. Now with the help of this RNA primer DNA polymerase 3 enzyme which has 5 prime to 3 prime polymerase activity. So it can synthesize the DNA strand which is complementary to the template strand. And this DNA synthesis or DNA replication on the leading strand it. occurs from 5'to 3'direction by the activity of enzyme DNA polymerase 3. It also has 3'to 5'exonuclease activity which is required for proofreading or error detection and correction. So, this is how elongation of leading strand which is continuous, it occurs from 5'to 3'direction with the help of enzyme DNA polymerase 3. Elongation of lagging strand is different from elongation of leading strand. Elongation of lagging strand is semi-discontinuous and it occurs with the help of synthesis of short stretches of DNA which are called as Okazaki fragments. So for this synthesis of DNA or elongation of lagging strand requires RNA primer and this RNA primer is synthesized by DNA primase which is also called as DNAG. So this is how RNA primers are synthesized on the lagging strand. Now after the synthesis of RNA primer now DNA polymerase 3 it can catalyze polymerization by its 5'to 3'polymerase activity. So this DNA polymerase then synthesize a short stretch of DNA then it gets dissociated and again it binds to the other site and there is formation or synthesis of other short stage of DNA and all these short stages of newly synthesized DNA they are called as Okazaki fragments and the DNA replication or DNA synthesis on the lagging strand it is semi-discontinuous and away from the direction of replication 4. So this DNA polymerase 3 it has 5 prime to 3 prime polymerase activity which helps in the synthesis of DNA on lagging strand. that is the okazaki fragments and the proofreading activity is provided by 3 prime to 5 prime exonuclease activity of dna polymerase 3. on the lagging strand dna polymerase 3 is responsible for synthesis of short stretches of dna which are called as okazaki fragment now after the activity of dna polymerase 3 dna polymerase 1 comes into picture and it has three types of activity so five prime to three prime exonuclease activity of dna polymerase one is responsible for removal of primer so primer on the lagging strand they are removed with the help of five prime to three prime exonuclease activity of dna polymerase one now gap is created between different okazaki fragments so this gap is filled by five prime to three prime polymerase activity of dna polymerase one and It also has 3'to 5'exonuclease activity for proofreading i.e. error detection and correction. But there is also gap between DNA synthesized by DNA polymerase 3 and the gap which is filled by DNA polymerase 1. So ultimately the ends are not joined. So this joining of ends it is caused by DNA ligase. So this DNA ligase enzyme. It causes final phosphodiester linkage formation between DNA synthesized by DNA polymerase 3 and by DNA polymerase 1. So by the activity of this DNA ligase and finally this DNA strand is synthesized from 5'to 3'direction on the ligand strand. The third and last stage of process of replication of DNA is termination and this termination region is present in the DNA template which has multiple copies of 20 base pair sequences. As the moving replication fork encounters this sequence, its further movement is arrested and other replication fork coming from the other direction in circular DNA encounters the first fork and further replication stops and that's how DNA replication occurs. Let's summarize today's topic of DNA replication. So replication is the process of synthesis of identical duplicate copy of DNA from an existing DNA molecule. And this replication is semi-conservative. It is bidirectional and this process of replication is accurate. It proceeds from 5'to 3'direction and both strands of DNA can act as a template strand. And it requires various factors like parent DNA. nucleotides that is ribonucleotides for RNA synthesis, deoxyribonucleotides for DNA polymerization and various enzymes and proteins are required. So the first this process of DNA replication requires various DNA proteins like DNA A which is the initiator protein which initiates this process then the enzyme helicase which is also called as DNA B which is responsible for separation of two complementary DNA strand and it leads to formation of replication bubble and replication fork and the other enzymes which are involved in the process of replication are topoisomerases which relieve the supercoiling in the dna which is formed due to separation of strands there are single standard binding proteins which bind the separated dna strand and prevent their re-annelling the enzyme primase dna primase which is also called as dna g it is required for RNA primer synthesis. So this RNA primer synthesis is very important because this RNA primer synthesis initiates DNA replication because DNA polymerase enzyme cannot directly initiate the process of replication. Two important enzymes, polymerase enzymes are important DNA polymerase 3 and 1. DNA polymerase 3 is responsible for chain elongation in 5'to 3'direction on both leading strand as well as lagging strand and it also has proofreading activity and DNA polymerase 1 is responsible for excision of primer gap filling as well as proofreading activity and lastly DNA ligase this DNA ligase enzyme It catalyzes the final phosphodiester linkage between DNA synthesized by DNA polymerase 3 and by DNA polymerase 1 on the lagging strand. The stages of DNA replication process includes initiation, elongation and termination and this initiation process includes separation of DNA strand at Oric and this DNA is an initiated protein which binds to this Oric region and there is formation of replication bubble and various proteins and enzymes they play important role in formation of this replication bubble. Then comes The elongation. Elongation on leading strand involves primer synthesis with the help of enzyme DNA primase that is DNAG and then DNA polymerase 3 causes DNA polymerization on the leading strand which is continuous in 5 prime to 3 prime direction. While on the lagging strand this elongation involves primer synthesis by enzyme primase DNAG then DNA polymerization which is discontinuous it occurs with the help of DNA polymerase 3 and the short stretches of DNA are synthesized which are called as Okazaki fragments. Then there is excision of primer which is carried out by DNA polymerase 1 and then the gaps are filled by DNA polymerase 1 it has polymerase activity and joining of ends of DNA which are synthesized by DNA polymerase 3 and 1 on this lagging stand it is caused by by DNA ligase and finally the termination occurs and that's how the process of replication occurs in E. coli. So I hope this video will help you to understand the basics of DNA replication in E. coli. So thank you for watching.

So the first process is replication. In this process of replication, the parental DNA is copied to form daughter DNA molecule with identical nucleotide sequence. So the first process of flow of genetic information is replication. The second is transcription in which the genetic information contained in the DNA is transferred into RNA.

And the third is translation whereby the genetic message encoded in RNA is translated on ribosomes into a polypeptide with a specific amino acid sequence. So by these three processes a gene which is the fundamental unit of genetic information it is used to produce a unique functional molecule and this functional molecule is usually protein or RNA. DNA replication is essential for the transmission of genomes and the genes they contain from one generation to other generation.

It is required for continuation of species and occurs during cell division. Replication is very well studied in prokaryotes like E. coli. So what is replication?

It is the process of synthesis of an identical duplicate copy of DNA from an existing DNA molecule. And DNA is replicated only once during the life cycle of a cell. So this is a duplex DNA strand from which two identical DNA stands are formed and this process is called as replication and the newly synthesized DNA stand there is one stand is newly synthesized and one stand is a parental stand and this replication is semi-conservative.

Why this is called semi-conservative? It refers to the duplication of double standard DNA molecule in a such a way that the new DNA molecule has one original parental stand and one newly synthesized stand. This New double stranded DNA is identical to the original parental duplex DNA molecule.

And this semi-conservative replication, it was proposed by Watson and Oric after they proposed the double helix model of DNA structure. Now let's see some salient features of replication. Each of the two DNA strand is used as a template to synthesize a new complementary strand to it. And new DNA is synthesized in 5'to 3'direction and for this the template strand is read from 3'to 5'end.

So the synthesis of DNA occurs from 5'to 3'direction on both the template strands. Replication begins at origin and it is the specific site having 245 base pairs and it is called as ORIC. And at this particular site, when DNA untwist to expose the two single template strands for DNA replication, a Y-shaped structure is formed.

So because of separation of two DNA, Y-shaped structure is formed and it is called as replication fork. And this replication fork, it moves in the direction of untwisting the DNA. So replication fork is moving.

in both the direction and that's why the replication is bidirectional. So when DNA untwist, there is formation of two replication fork and these two Y shaped structure join together at their tops to form replication bubble. So this is called as replication bubble and it is nothing but the locally denatured segment of DNA.

into one of the replication fork and this is the direction of movement of replication fork. Now let's understand what does it mean by leading strand and lagging strand. So on this strand, this upper strand, the DNA synthesis or DNA replication occurs from 5 prime to 3 prime direction in continuous manner. So this is called as leading strand. On the leading strand, replication occurs continuously in continuous manner.

from 5 prime to 3 prime direction and also in the direction of movement of replication fork. So this is leading strand. Now what is lagging strand? The other one is lagging strand. DNA does not, synthesis does not occur here continuously.

It occurs in short stretches. Short stretches of DNA are synthesized. So here the replication is not continuous. it is discontinuous but from 5'to 3'direction and this strand is called as lagging strand. And these short stretches of newly synthesized DNA they are called as Okazaki fragments.

So here on the lagging strand replication is discontinuous and away from the direction of moment of replication 4. Though it is from 5'to 3'but it is away or opposite in the direction of replication fork while in case of leading strand replication is continuous and in the same direction of movement of replication fork. Though on the leading strand replication is continuous and on the lagging strand replication is discontinuous but it occurs simultaneously. For replication various factors are required and one of the factor is parental DNA as a template. Second factor required for replication is nucleotides, ribonucleotides for RNA primer synthesis. So these are various ribonucleotides having bases AUGC.

Deoxyribonucleotides are required for complementary DNA strand synthesis. These are various deoxyribonucleotides having bases ATGC and various enzymes and accessory proteins are required for replication. So let's just enumerate various enzymes and proteins required for replication.

We will see functions in later slides. So the various proteins and enzymes required are DNA A, helicases which is also called as DNA B and DNA C. These are required for DNA replication. Topoisomerases, these enzymes topoisomerase 1 and topoisomerase 2 are also required. Single standard binding protein are required. Primase.

which is also called as DNAG it is required for RNA primer synthesis then DNA in DNA replication DNA polymerase 1 and 3 play very important role DNA ligase so these are various enzymes and proteins which are required for DNA replication DNA replication enzymes are called as DNA polymerase and this DNA polymerase catalyze replication but it requires a small piece of RNA it is called as primer. So RNA primer initiates DNA synthesis. So this is the primer which is 10 to 16 nucleotides long and it has 3 3 prime hydroxyl group and the DNA polymerase can add nucleotides to this 3 3 prime hydroxyl group of this RNA primer to replicate the DNA because DNA polymerase cannot add nucleotide without a primer.

Now how this primer is synthesized? This RNA primer it is synthesized by enzyme called as DNA primase. This is also called as DNAG.

It is modified RNA polymerase and it forms RNA primer. And on this RNA primer then DNA polymerase 3 can add new nucleotides on this RNA primer. So The new deoxyribonucleotide triphosphates, it is linked to free 3'hydroxyl end of the ribose and the nucleotides are joined to one another by 3'5'phosphodiester bond.

There is release of pyrophosphate. So the energy required for this process of adding nucleotides, it is provided by this release of pyrophosphate and this is how DNA polymerase can add or it can catalyze the polymerization of various nucleotide precursors into DNA chain and the direction of synthesis of new DNA chain is from 5 prime to 3 prime because DNA polymerase can add new nucleotide at 3 prime end of the previous ribose sugar or deoxyribose sugar and that's why DNA synthesis occurs from 5 prime to 3 prime direction. Now at each step of lengthening of new DNA chain, DNA polymerase finds the correct precursor that is deoxyribonucleotide triphosphate that can form a complementary base pair with the nucleotide on the template strand of DNA.

So RNA primers initiates DNA synthesis and without RNA primer, DNA polymerase cannot initiate or cannot replicate the DNA. There are 5 DNA polymerases known in E.coli and out of these 5, DNA polymerases 1, 2 and 3 they have role in DNA replication while type 4 and 5 they have role in DNA repair mechanisms. So DNA polymerase 1, 2 and 3 all have 3'to 5'exonuclease or proofreading activity while only DNA polymerase 1 it has 5'to 3'exonuclease activity. The polymerization rate that is the number of nucleotides added per second during replication is highest for DNA polymerase 3 that is 250 to 1000 nucleotides per second are added. Processivity it refers to number of nucleotides added by the enzyme before it dissociates from the DNA template and that processivity is also means highest for DNA polymerase 3 leading to the fastest chain elongation and this DNA polymerase 3. It is a multimeric enzyme with more than 10 subunits and two of its subunits are called as beta subunits and they act like a sliding clamp enhancing its stability and processivity.

So the main role of DNA polymerase 1 it is the gap filling, main role of DNA polymerase 2 is repair, DNA repair and main role of DNA polymerase 3 it is the DNA replication. DNA replication is accurate because of DNA proofreading mechanism. And DNA polymerase 3 and DNA polymerase 1, they both have 3'to 5'exonuclease activity. It means that they can remove nucleotides from 3'end of DNA chain and it is useful for proofreading i.e. error correction.

If an incorrect base is inserted by DNA polymerase, which can be 1 base in million, can be incorrect. So this error is recognized immediately by the enzyme and the enzyme 3'to 5'exonuclease activity excises the eraneous nucleotide from the new strand and then the DNA polymerase resume forward movement and inserts the correct nucleotide. In addition to this DNA polymerase 1 also has 5'to 3'exonuclease activity and it can remove nucleotides from the 5'end of DNA.

So far we have studied various salient features of replication. So replication is semi-conservative, it is simultaneous and it is bidirectional. Replication is accurate because of proofreading mechanism due to 3'to 5'exonuclease activity of DNA polymerase 3 and 1. There is a formation of replication fork and replication begins at the origin and there is also formation of replication bubble in the process of replication and various enzymes and proteins are required for the replication along with various nucleotides, ribonucleotides and deoxyribonucleotides and this process of replication is catalyzed by DNA polymerases 3 and 1 but This DNA polymerase require RNA primer which is synthesized by enzyme primase and without which DNA cannot polymerize or cannot replicate the DNA. So these are the various salient features.

Now let's understand the process of replication. Replication occurs in three stages and the first stage is initiation of replication. Second stage is elongation of replication. And third is termination of replication. Let's begin with the initiation of replication.

And the initiation of replication is directed by a DNA sequence. It is called as replicator. And this replicator includes origin of replication where this replication begins. This is the area of origin of replication called as Oric.

And it has two 45 specific base pairs. And this is the specific region where double. DNA double helix denatures into single strands within which replication begins.

So this is the origin of replication. This ORIC where initiation begins it is 245 base pair long and it has two types of repeated conserved sequences. So there are three repeats of 13 base pair sequences reaching A and T bases and five repeats of nine base pair sequences.

So there are two types of sequences 13 base pair repeats and 9 base pair repeats and one important protein that is DNA A protein along with ATP molecule it binds to this 9BP repeats and by binding to this 9BP repeats after that it recognizes and denatures this 13BP region which is reaching A and T bases so that's how this DNA protein help in the initiation of replication. The recognition and denaturation of 13 base pair region by DNA protein causes separation of DNA strand and there is formation of replication bubble and replication fork. DNA is an initiator protein which identifies Oric and causes separation of two strands by denaturation which allows binding of several other proteins like helicases, primase and polymerases. So helicase B which is also called as DNA B it is involved in separation of DNA strand and this helicases are recruited and loaded onto DNA strand by other protein which is called as DNA C. It is also called as DNA helicase loader protein. And this helicases untwist the DNA by breaking hydrogen bonds between the bases and the energy for untwisting comes from hydrolysis of ATP.

So helicase requires ATP. This helicase and DNA-C both are hexameric string like molecules and each strand require one helicase molecule. So both strands require two helicase.

DNA-C will help this helicase to load on this DNA strand. So this helicase molecule it will separate two strands of DNA. Now single standard binding protein.

They bind. to the separated strand and prevent their re-naturation. So the separated strands are now they are stabilized for replication.

So single standard binding protein they prevent re-annelling of the separated strand. Now the another enzyme topoisomerase. So this topoisomerase there are two types.

Type 1 it reversibly cut one strand of DNA It has both nucleus and ligase activity and it does not require ATP. And the other type is DNA gyrase which make transient breaks in both the strand and it requires ATP. What is the function of this topoisomerase?

So this topoisomerase relieves the supercoiling developed in DNA due to separation of strands. So it relieves supercoiling by creating NIC in the DNA strand. and by its nucleus and ligase activity then it can repair that DNA.

So this process of initiation starts from the DNA A protein which identifies Oric then comes the role of DNA B and DNA C. DNA C will help this DNA B to get loaded on the DNA strand then this helicase will separate two DNA strands. The single-stranded binding protein will bind to the separated DNA strand and prevent its re-annulling. The enzyme topoisomerase binds to the DNA and relieves supercoiling which is developed in the DNA due to separation of strands.

So after initiation of this replication, the elongation begins. This elongation is synthesis of a new strand of DNA using two separated strands as the template. New bases are added according to the principle of complementary base pairing depending on the sequence of bases in the template strand.

So the nucleotide A in template strand is paired with T in the new strand. Similarly G is paired with C. So the replication occurs differently in two strands which are called as leading strand and lagging strand.

So on the leading strand replication is continuous while on the lagging strand the replication is discontinuous. So leading strand runs from 3'to 5'direction and replication occurs in 5'to 3'direction. So for process of this elongation we require the enzyme DNA primase which is called as DNAG and it is required for synthesis of RNA primer because RNA primer initiates DNA synthesis.

DNA polymerase cannot start this process of replication it requires RNA prime. So this is a DNA primase DNA G and it is required for synthesis of RNA primer. Now with the help of this RNA primer DNA polymerase 3 enzyme which has 5 prime to 3 prime polymerase activity.

So it can synthesize the DNA strand which is complementary to the template strand. And this DNA synthesis or DNA replication on the leading strand it. occurs from 5'to 3'direction by the activity of enzyme DNA polymerase 3. It also has 3'to 5'exonuclease activity which is required for proofreading or error detection and correction.

So, this is how elongation of leading strand which is continuous, it occurs from 5'to 3'direction with the help of enzyme DNA polymerase 3. Elongation of lagging strand is different from elongation of leading strand. Elongation of lagging strand is semi-discontinuous and it occurs with the help of synthesis of short stretches of DNA which are called as Okazaki fragments. So for this synthesis of DNA or elongation of lagging strand requires RNA primer and this RNA primer is synthesized by DNA primase which is also called as DNAG. So this is how RNA primers are synthesized on the lagging strand.

Now after the synthesis of RNA primer now DNA polymerase 3 it can catalyze polymerization by its 5'to 3'polymerase activity. So this DNA polymerase then synthesize a short stretch of DNA then it gets dissociated and again it binds to the other site and there is formation or synthesis of other short stage of DNA and all these short stages of newly synthesized DNA they are called as Okazaki fragments and the DNA replication or DNA synthesis on the lagging strand it is semi-discontinuous and away from the direction of replication 4. So this DNA polymerase 3 it has 5 prime to 3 prime polymerase activity which helps in the synthesis of DNA on lagging strand. that is the okazaki fragments and the proofreading activity is provided by 3 prime to 5 prime exonuclease activity of dna polymerase 3. on the lagging strand dna polymerase 3 is responsible for synthesis of short stretches of dna which are called as okazaki fragment now after the activity of dna polymerase 3 dna polymerase 1 comes into picture and it has three types of activity so five prime to three prime exonuclease activity of dna polymerase one is responsible for removal of primer so primer on the lagging strand they are removed with the help of five prime to three prime exonuclease activity of dna polymerase one now gap is created between different okazaki fragments so this gap is filled by five prime to three prime polymerase activity of dna polymerase one and It also has 3'to 5'exonuclease activity for proofreading i.e. error detection and correction.

But there is also gap between DNA synthesized by DNA polymerase 3 and the gap which is filled by DNA polymerase 1. So ultimately the ends are not joined. So this joining of ends it is caused by DNA ligase. So this DNA ligase enzyme.

It causes final phosphodiester linkage formation between DNA synthesized by DNA polymerase 3 and by DNA polymerase 1. So by the activity of this DNA ligase and finally this DNA strand is synthesized from 5'to 3'direction on the ligand strand. The third and last stage of process of replication of DNA is termination and this termination region is present in the DNA template which has multiple copies of 20 base pair sequences. As the moving replication fork encounters this sequence, its further movement is arrested and other replication fork coming from the other direction in circular DNA encounters the first fork and further replication stops and that's how DNA replication occurs.

Let's summarize today's topic of DNA replication. So replication is the process of synthesis of identical duplicate copy of DNA from an existing DNA molecule. And this replication is semi-conservative.

It is bidirectional and this process of replication is accurate. It proceeds from 5'to 3'direction and both strands of DNA can act as a template strand. And it requires various factors like parent DNA. nucleotides that is ribonucleotides for RNA synthesis, deoxyribonucleotides for DNA polymerization and various enzymes and proteins are required.

So the first this process of DNA replication requires various DNA proteins like DNA A which is the initiator protein which initiates this process then the enzyme helicase which is also called as DNA B which is responsible for separation of two complementary DNA strand and it leads to formation of replication bubble and replication fork and the other enzymes which are involved in the process of replication are topoisomerases which relieve the supercoiling in the dna which is formed due to separation of strands there are single standard binding proteins which bind the separated dna strand and prevent their re-annelling the enzyme primase dna primase which is also called as dna g it is required for RNA primer synthesis. So this RNA primer synthesis is very important because this RNA primer synthesis initiates DNA replication because DNA polymerase enzyme cannot directly initiate the process of replication. Two important enzymes, polymerase enzymes are important DNA polymerase 3 and 1. DNA polymerase 3 is responsible for chain elongation in 5'to 3'direction on both leading strand as well as lagging strand and it also has proofreading activity and DNA polymerase 1 is responsible for excision of primer gap filling as well as proofreading activity and lastly DNA ligase this DNA ligase enzyme It catalyzes the final phosphodiester linkage between DNA synthesized by DNA polymerase 3 and by DNA polymerase 1 on the lagging strand.

The stages of DNA replication process includes initiation, elongation and termination and this initiation process includes separation of DNA strand at Oric and this DNA is an initiated protein which binds to this Oric region and there is formation of replication bubble and various proteins and enzymes they play important role in formation of this replication bubble. Then comes The elongation. Elongation on leading strand involves primer synthesis with the help of enzyme DNA primase that is DNAG and then DNA polymerase 3 causes DNA polymerization on the leading strand which is continuous in 5 prime to 3 prime direction. While on the lagging strand this elongation involves primer synthesis by enzyme primase DNAG then DNA polymerization which is discontinuous it occurs with the help of DNA polymerase 3 and the short stretches of DNA are synthesized which are called as Okazaki fragments.

Then there is excision of primer which is carried out by DNA polymerase 1 and then the gaps are filled by DNA polymerase 1 it has polymerase activity and joining of ends of DNA which are synthesized by DNA polymerase 3 and 1 on this lagging stand it is caused by by DNA ligase and finally the termination occurs and that's how the process of replication occurs in E. coli. So I hope this video will help you to understand the basics of DNA replication in E. coli. So thank you for watching.

Transcript for:Prokaryotic DNA Replication Overview

Transcript for:
Prokaryotic DNA Replication Overview