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DNA Replication And Primer


Why does nature rely on RNA primer for the start of DNA Replication? Why not simply use DNA primer and make life simple !


Biochemically none of the DNA-dependent DNA polymerases involved in DNA replication have the ability to begin elongation without a 5' to 3' primer.

The only DNA Polymerase that can catalyze elongation without a 5' to 3' primer is Reverse Transcriptase, however Reverse Transcriptase is an RNA-dependent DNA polymerase, and that accounts for the difference.

And if your next question is, but why, the answer is that that is how the systems evolved. Also DNA Polymerases have a much higher rate of fidelity due to error checking than RNA Polymerases do, and as the RNA primers get removed anyway and replaced with DNA, an error here or there in the primer will not matter.

The RNA-DNA duplex is also less stable than the DNA to DNA duplex, so it makes it easier for FEN1 to remove an RNA Primer than it would a DNA primer, assuming that the mechanism of primer removal and replacement after the complementary leading and lagging strands have been synthesized remained in place.


DNA Replication And Primer - Biology

The formation of carbon copy or replica of D.N.A from parental D.N.A is called replication of D.N.A or duplication of D.N.A.The replication takes place inside the nucleus of eukaryotes in the S-phage of the cell cycle and in the cytoplasm of the prokaryotic cell. Watson and Crick not only proposed the three-dimensional model of D.N.A but also suggested the mechanism of D.N.A replication. In replication, both D.N.A strands uncoil and separate. Each strand acts as a template strand for the formation of a new and complementary D.N.A strand. The template and newly synthesized strand form a new double strand which is identical to the parent D.N.A molecule.

Methods of Replication:

Del Bruck suggested three theoretical methods for the replication of Watson and Crick model of D.N.A. They are as follows :

1)Conservative mode of replication:-

In this type of replication, double-stranded parent D.N.A synthesizes replica or new D.N.A and then it remains conserved as it is.

2) Dispersive mode of replication:-

In this type of replication, the parental D.N.A synthesizes two daughters D.N.A after which it(parental D.N.A) gets disintegrated.

3)Semi-conservative mode of replication:-

Meselson and Stahl by using N 14 and N 15 isotopes of Nitrogen confirmed that the replication of D.N.A inE.coli bacterium is semi-conservative.

In this method, two strands are separated by breaking hydrogen bonds of their nitrogen bases. Both the strands of DNA function as master or template strand which synthesize new or complementary strands from the medium. As a result, two new DNA molecules are synthesized in which one strand is original and another strand and the other strand is newly assembled. Thus, a newly synthesized DNA is also called hybrid DNA.

Basic requirements for DNA replication:

The mechanism of DNA replication is a complex and involves many steps. Several enzymes, protein factors and metal ions are required for this process.

A) DNA helicase: It is an enzyme which breaks the H2 bonds and separates the DNA strands. Thus, a fork is formed at the junction known as replication fork.

B)Single-Stranded Binding (SSB) proteins:These are the protein molecules which attach tightly to the exposed single-stranded DNA in order to stabilize the single-stranded DNA long enough for replication.

C)Primase:It is an enzyme responsible for the synthesis of short RNA primers. AnRNA primer is a small strand of RNA which guides the replication.

D)DNA polymerase:It is an enzyme responsible for catalyzing the synthesis of DNA. It can add nucleotides to the template DNA,

E)Topoisomerase:It is an enzyme responsible for causing nick(cut) in the DNA strand so as to release the tension created during unwinding of DNA.

F)RNAse:It is an enzyme which digests the RNA primers after DNA synthesis is over.

G)DNA ligase:It is an enzyme which seals the gaps in the synthesized DNA strand.

H)Substrates:The four deoxyribonucleoside triphosphates(dNTPs) such as dATPs, dGTPs, dCTPS, and dTTPS are needed as substrates for DNA synthesis. Cleavage of the high-energy bond between phosphates provides the energy for the addition of the nucleotide.

I)Folic acid:It is essential for the synthesis of nitrogen bases.

J)Mg 2+ and Mn 2+ ions:These are essential for DNA synthesis.

Process of semi-conservative mode of replication of DNA:

The mechanism of DNA replication is a complex and involves many steps. The steps are:

1) Initiation of DNA replication

2) Activation of deoxyribonucleotides

3) Exposure of DNA strands or nitrogen bases and formation of Y-shaped fork

6) Formation of new DNA strands

7)Proof-reading and DNA repair

8)Termination of DNA helix formation.

1)Initiation of DNA replication-

The point in the DNA from where replication begins is called initiation point or origin of replication or ori-site. It is formed by the breakage or nicking of phosphodiester bond by the enzyme endonuclease without the removal of nucleotides.

In the prokaryotic cell, DNA forms a single origin of replication and in the eukaryotic cell, DNA has many ori-sites.

2) Activation of deoxyribonucleotides-

Four types of inactive deoxyribonucleoside monophosphates of DNA i:e deAMP, deGMP, deCMP, and deTMP are found in the nucleoplasm react with ATP molecules in the presence of enzymes phosphorylase to form four types of deoxyribonucleotide triphosphates of DNA i:e deATP, deGTP, deCTP, and deTTP. This process is called phosphorylation. [ip=inorganic phosphate]

fig: Activation of deoxyribonucleotides

3)Exposure of DNA strands and formation of replication fork-

Enzyme helicase breaks the hydrogen bonds between the nitrogen bases of two strains of DNA at ori-site. As a result, the two strands of DNA are separated from each other and are in a state of supercoiling tension. To relief from this tension, enzyme topo-isomerase-I or DNA gyrase plays a vital role. The separated strands are established in the Y-shaped structure called replication fork held by single-stranded binding(SSB) protein.

4) Formation of RNA primer-

DNA replication always takes place in 5' to 3' direction on the master or template strands. TO initiate the DNA synthesis, a short sequence of RNA primer is required. RNA primer is synthesized by an enzyme DNA polymerase, called primase. RNA primer is formed on the free end of one DNA strand (3' end) and another in the fork end of another master strand of DNA.

5) Base pairing-

The separated DNA strands are known as templates. The active deoxyribonucleotides present in the nucleoplasm comes to lie opposite to the specific nitrogen bases of both the strand of DNA and bind together according to base pairing rule i:e A=T, G&equivC, and T=A, C&equivG. With the help of enzyme pyrophosphatase, two extra phosphates present in the deoxyribonucleotide triphosphate separate and energy is released. This energy is used in the formation of hydrogen bonds between the free nucleotides of nucleoplasm and nucleotides of master strands.

deoxyribonucleotide triphosphate (in the presence of pyrophosphatase enzyme) &rarr deoxyribonucleoside monophosphate +2ip

deATP(in presence of pyrophosphatase enzyme)&rarr deAMP + 2ip + energy

6) Formation of new DNA strands-

DNA polymerase enzyme, energy-rich molecule ATP along with manganese and magnesium ion are responsible for the formation of new DNA strands. New DNA strand is synthesized continuously in 5' to 3' direction on 3' to 5' master strand which is called leading strand.

On the other hand, discontinuous fragments of DNA are also formed in 5' to 3' direction of the other master strand which is called lagging strand. The discontinuous fragments formed by each RNA primer are called Okazaki fragments.

7) Proof-reading and DNA repair-

During replication, the accuracy of base pairing is essential. Sometimes, wrong bases do get in the new strand. The frequency of this introduction of a base is in 100,000. These are noted and removed by the exo-nucleus activity of DNA polymerase-I enzyme (proof-reader). The newly formed DNAstructuree is repaired by DNA ligase enzyme.

8) Termination of DNA helix formation-

Generally, DNA replication stops when two replicating forks meet to each other. But in prokaryotes, a terminating protein called 'Tus' prevents the movement of helicase and indicates the completion of the replication process.

Thus, replication of DNA is a semi-conservative process because parental DNA replicates and forms two daughter DNAs in which one strand of them is parental and another is newly formed.

source: www.rothamsted.ac.uk fig: semi-conservative mode of DNA replication.

Keshari, Arvind K. and Kamal K. Adhikari. A Text Book of Higher Secondary Biology(Class XII). 1st. Kathmandu: Vidyarthi Pustak Bhandar, 2015.

Mehta, Krishna Ram. Principle of biology. 2nd edition. Kathmandu: Asmita, 2068,2069.


RNA primer:

First of all, the RNA primer is non-significant for the PCR amplification. However, it is necessary for the replication process.

The RNA primer is a short stretch of nucleic acid made up of the single-stranded RNA molecule.

An RNA polymerase, called DNA primase synthesizes a short stretch of single-stranded RNA molecule for starting replication.

It is very essentially required for a DNA polymerase to start its catalytic activity.

The single-stranded RNA primer provides a free 3’ OH group which is required for DNA polymerase. See the Image,

The RNA primers are of two types used in replication. One, that starts DNA replications and is approximately 10 to 18 nucleotides long, at the leading strand.

While others are used for the synthesis of Okazaki fragments and these are 8 to 10 nucleotides long, at the lagging strand.

Why replication is dependent on RNA primer instead of a DNA primer?

The only RNA polymerase is available to synthesize the ssRNA thus not DNA but the RNA is used in the replication.

And in the end, due to the exonuclease activity of the DNA polymerase, the RNA primers are removed, simultaneously the gap is filled by the polymerase with the complementary nucleotides and sealed with DNA ligase.

For doing this, the DNA polymerase trackback and finds the RNA primer which is actually not a part of our DNA strand.

(DNA ligase fills the gap between adjacent nucleotides by forming the phosphodiester bond.)

The 3’ to 5’ exonuclease activity of DNA polymerase removes it.

Note: only a single type of RNA primer is used for DNA replication.

Interesting fact:

The DNA polymerase can elongate the polynucleotide strand but can not synthesise it directly (it needs free 3’ end).

Only RNA polymerase can do so, thus, RNA primer is used in replication.

There is a difference between synthesizing and elongating nucleotide chains, see the image below,


Initiation of DNA Replication: Preparatory Step

Step 1: Replication fork formation.

Before DNA can be replicated, the double-stranded molecule must be “unzipped” into two single strands. As we know, DNA has four bases called adenine (A), thymine (T), cytosine (C), and guanine (G) that form pairs between the two strands. Adenine only pairs with thymine and cytosine only binds to guanine.

In order to unwind DNA, these base-pair interactions must be broken. This is done by an enzyme known as DNA helicase.

However, there is a special initiator protein that is required to trigger DNA replication, namely DnaA. It binds regions at the oriC site throughout the cell cycle. In order to initiate the replication, however, the DnaA protein must bind to a few specific oriC sequences that have five repeats of the 9 bp sequence (also known as the R site).

When DnaA binds to the oriC site, it recruits a helicase enzyme (DnaB helicase). Now the DNA helicase breaks the hydrogen bond that holds complementary DNA bases together.

The separation of the two single strands of DNA creates a two Y-shaped structure called a replication fork. together they form a bubble-like structure called a replication bubble. These two separate strands serve as a template for the production of the new DNA strands.

How does the replication actually work on the replication forks?

Helicase is the first replication enzyme to be loaded at the origin of replication. Helicase’s job is to simply move the replication forks forward by “unwinding” the DNA. As we know, DNA is very unstable in the form of a single strand. In this way, cells can prevent them from coming back together in a double helix.

To do this, a specific protein called single-stranded DNA binding proteins (SSBs) coats and keeps the separated strands of DNA near the replication fork.

When the helicase quickly unwinds the double helix. It increases the tension on the remaining DNA molecule. Topoisomerase plays an important maintenance role during DNA replication. This enzyme prevents the DNA double helix in front of the replication fork from becoming too tight when the DNA is opened. It does this by making temporary nicks in the helix to release tension and then sealing the nicks to prevent permanent damage.


POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA

Mitochondria are vital in providing cellular energy via their oxidative phosphorylation system, which requires the coordinated expression of genes encoded by both the nuclear and mitochondrial genomes (mtDNA). Transcription of the circular mammalian mtDNA depends on a single mitochondrial RNA polymerase (POLRMT). Although the transcription initiation process is well understood, it is debated whether POLRMT also serves as the primase for the initiation of mtDNA replication. In the nucleus, the RNA polymerases needed for gene expression have no such role. Conditional knockout of Polrmt in the heart results in severe mitochondrial dysfunction causing dilated cardiomyopathy in young mice. We further studied the molecular consequences of different expression levels of POLRMT and found that POLRMT is essential for primer synthesis to initiate mtDNA replication in vivo. Furthermore, transcription initiation for primer formation has priority over gene expression. Surprisingly, mitochondrial transcription factor A (TFAM) exists in an mtDNA-free pool in the Polrmt knockout mice. TFAM levels remain unchanged despite strong mtDNA depletion, and TFAM is thus protected from degradation of the AAA(+) Lon protease in the absence of POLRMT. Last, we report that mitochondrial transcription elongation factor may compensate for a partial depletion of POLRMT in heterozygous Polrmt knockout mice, indicating a direct regulatory role of this factor in transcription. In conclusion, we present in vivo evidence that POLRMT has a key regulatory role in the replication of mammalian mtDNA and is part of a transcriptional mechanism that provides a switch between primer formation for mtDNA replication and mitochondrial gene expression.

Keywords: 7S RNA Mitochondria POLRMT knockout mouse light strand promoter mitochondrial RNA polymerase mitochondrial gene expression mtDNA mtDNA replication mtDNA-free TFAM pool twinkle.

Figures

Fig. 1. Knockout of Polrmt in germline…

Fig. 1. Knockout of Polrmt in germline and heart.

( A ) RT-PCR analysis of…

Fig. 2. Reduced OXPHOS capacity in Polrmt…

Fig. 2. Reduced OXPHOS capacity in Polrmt knockout mouse heart.

( A ) Transmission electron…

Fig. 3. Decreased mtDNA replication in Polrmt…

Fig. 3. Decreased mtDNA replication in Polrmt knockout mice.

Fig. 4. Loss of POLRMT results in…

Fig. 4. Loss of POLRMT results in an increased mtDNA-free pool of TFAM.

Fig. 5. LSP and HSP show different…

Fig. 5. LSP and HSP show different sensitivities at low POLRMT concentrations.

Fig. 6. Characterization of heterozygous Polrmt knockout…

Fig. 6. Characterization of heterozygous Polrmt knockout mice.

( A ) POLRMT steady-state protein levels…

Fig. 7. Model of POLRMT regulating replication…

Fig. 7. Model of POLRMT regulating replication primer formation and expression of mtDNA.


Primase

Abstract

Primase is the enzyme that synthesizes RNA primers, oligonucleotides that are complementarily bound to a nucleic acid polymer. Primase is required because DNA polymerases cannot initiate polymer synthesis on single-stranded DNA templates they can only elongate from the 3′-hydroxyl of a primer. Primases fall into two major sequence and structure families: bacterial and archaeal/eukaryotic nuclear. Bacterial primases are monomers consisting of three domains. The N-terminal domain has a zinc-finger motif and is likely responsible for the initiation specificity of this enzyme. The central catalytic domain binds single-stranded DNA and catalyzes RNA polymer initiation and elongation complementary to it. The C-terminal domain interacts with other proteins, including DnaB helicase so that its activity takes place at the replication fork. The bacterial primase gene, dnaG, is the central gene of the macromolecular synthesis operon carrying the genes for the initiation phases of translation, replication, and transcription. Of the three genes, dnaG is under the most levels of control and is expressed in the lowest amount. Archaeal/eukaryotic primase resides in a heterotetramer consisting of a small primase subunit, a large primase subunit, a regulatory phosphoprotein, and DNA polymerase alpha. The small subunit has primer synthesis activity that is modulated by the other three proteins in the complex as well as by Replication protein A, a single-stranded DNA-binding protein required for lagging strand DNA synthesis, and the GINS complex, the central hub around which the leading- and lagging-strand DNA replicases assemble to control the progression of the replication fork. GINS interacts with the MCM helicase that translocates on the leading-strand template and also interacts with the DNA polymerase alpha/primase complex on the lagging strand.


DNA Replication: Introduction

DNA replication is the process of producing two identical replicas from one original DNA molecule. This biological process occurs in all living organisms and is the basis for biological inheritance. DNA is made up of two strands and each strand of the original DNA molecule serves as template for the production of the complementary strand, a process referred to as semiconservative replication. Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication.

For the DNA synthesis two key substrate is required.

Substrate: The four deoxynucleoside triphosphates (dNTPs)—deoxyadenosine tri- phosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphos-phate (dCTP), and deoxythymidine triphosphate (dTTP)—are needed as substrates for DNA synthesis.

Primer:template junction: second substrate for the DNA Synthesis is a particular arrangement of ssDNA and dsDNA called primer:template junction.

  • Template. DNA replication cannot occur without a template. A template is required to direct the addition of the appropriate complementary deoxynucleotide to the newly synthesized DNA strand. In semiconservative replication, each strand of parental DNA serves as a template. Then, each template strand and its newly synthesized complementary strand serve as the DNA in daughter cells.
  • Primer. DNA synthesis cannot start without a primer, which prepares the template strand for the addition of nucleotides. The primer must also have an exposed 3’OH because new nucleotides are added to the 3′ end of a primer that is properly base paired to the template strand of DNA, new synthesis is said to occur in a 5′ to 3′ direction.

The DNA synthesis that occurs during the process of replication is catalyzed by enzymes called DNA-dependent DNA polymerases. These enzymes depend on DNA to the extent that they require a DNA template. They are more commonly called DNA polymerases.


DNA Replication And Primer - Biology

DNA replication is a semi-conservative process that occurs during the S phase of interphase. It is the process by which an organism’s DNA is replicated to produce two identical copies.

It starts at the origin of replication. Prokaryotes have one origin of replication, whereas eukaryotes have multiple. At each origin of replication, there is a replication bubble that contains two replication forks. At each replication fork is the enzyme DNA helicase that unwinds the DNA’s double helix structure. It does this by breaking the hydrogen bonds between nitrogenous bases, separating the two DNA strands. Single strand binding proteins prevent separated strands from reattaching at the replication fork. The two separated strands of DNA are now called template strands.

DNA polymerase III is the enzyme that is used to build a complementary DNA strand using a template strand. It does this by attaching nucleoside triphosphates to the 3’ end of a nucleotide. DNA polymerase III also ensures that the nucleotides being attached have complementary bases to the template strand. However, DNA polymerase III cannot add nucleotides to the template strand. So the RNA primase creates RNA primers: short RNA sequences that are complementary to the DNA template strand. The DNA polymerase III then has available 3’ ends to add nucleotides to. However, DNA polymerase III can only do this in a 5’ to 3’ direction.

DNA is antiparallel that is, each strand goes in the opposite direction (as seen in the diagram below).

Because of this, the two template strands are also in different directions. In the diagram above, the strand on the right would be the leading strand. This means that after an RNA primer is placed at the 3’ end of this strand, DNA polymerase can build its complementary strand continuously in a 5’ to 3’ direction.

The strand on the right of the diagram is the lagging strand. The lagging strand appears to be growing in a 3’ to 5’ direction, but this is not the case. The lagging strand is divided into segments called Okazaki fragments that are being individually built in a 5’ to 3’ direction by DNA polymerase III. Each fragment begins with an RNA primer. As helicase unzips the DNA double helix, more and more primers are added.

Other enzymes are involved in DNA replication as well. DNA polymerase I removes RNA primers and replaces them with DNA nucleotides. DNA ligase forms phosphodiester bonds between the DNA nucleotides that DNA polymerase I adds and the ends of Okazaki fragments. Finally, enzymes proofread the DNA strands to check for mistakes, preventing mutation.

7.1.1 Nucleosomes help to supercoil the DNA.

Nucleosomes also help to regulate gene expression. Some DNA is wrapped around the histones in the nucleosome and is not accessible by the RNA polymerase so it cannot be transcribed

YouTube Video

7.1.2 DNA structure suggested a mechanism for DNA replication.

DNA is double stranded and the two strands are joined together via complementary base pairing. Therefore, it stands to reason that during replication the two strands separate and then through complementary base pairing nucleotides join the separated strands. And this would make 2 new, identical strands of DNA.

7.1.3 DNA polymerases can only add nucleotides to the 3’ end of a primer.

DNA polymerase cannot add bonds to the phosphate on the 5' end of DNA, so it creates bonds on the 3' end.

7.1.4 DNA replication is continuous on the leading strand and discontinuous on the lagging strand.

DNA replication occurs on both strand of the DNA, one strand is called the leading strand and the other is called the lagging. The leading strand is the one that moves in a 3' to 5' direction in the same way the helicase does.

7.1.5 DNA replication is carried out by a complex system of enzymes.

DNA polymerase III - Catalyzes the reaction that binds free floating nucleotides to make a new DNA strand

Helicase - Separates the original DNA strands

Gyrase/Topoisomerase - Helps uncoil the DNA helix

DNA Primase - Provides a site called the primase for the DNA polymerase III to begin adding nucleotides.

DNA Polymerase I - Replaces the RNA primer with DNA

Single Strand Binding Proteins - Prevents the DNA from re-annealing

Ligase - Joins the okazaki fragments together

7.1.6 Some regions of DNA do not code for proteins but have other important functions.

DNA that does not code for a protein is referred to as a non-coding sequence. Some non-coding sequences have important functions such as regulating gene expression by promoting and repressing the transcription of genes next to the.

Additionally, many DNA coding sequences are interrupted by non-coding sequences called introns. The introns are removed before translation but they are important in mRNA processing

On the ends of chromosomes are non-coding sequences called telomeres. During DNA replication the end of the molecule cannot be replicated so telomeres protect parts of the DNA from being lost during replication

Lastly, some non-coding sequences code for tRNA molecules instead of a protein.


7.1.7 Rosalind Franklin’s and Maurice Wilkins’ investigation of DNA structure by X-ray diffraction.

In 1950, Marice Wilkins developed a method of producing a way of imaging DNA molecule through X-Ray diffraction. Rosalind Franklin worked in the same lab as Wilkins and developed a high-resolution detector that took very clear images of DNA. Her findings were essential in the discovery of the double helix by Crick and Watson

7.1.8 Use of nucleotides containing dideoxyribonucleic acid to stop DNA replication in preparation of samples for base sequencing.

DNA sequencing is a process by which the order of the nucleotides in DNA can be found. One of the preliminary steps for DNA sequencing is fragmenting the DNA into pieces. To do this ddNA is added (dideoxyribonucleic acid) which do not have a OH molecule on the 3' end of the ribose sugar, this means that when the DNA polymerase reaches the ddNA it cannot continue replicating.

Gel electrophoresis is the process by which the sequence of the DNA is found. The fragmented DNA is put into a gel and an electric current runs through it. DNA is a polar molecule and is affected by the electric current and moves down the gel. Lighter/smaller fragments of the DNA moves further while heavier/longer fragments move very little. The result is a pattern of bands that one can use to figure out the sequence of the DNA. But remember that the sequence of the original strand is complementary the one shown by the banding patterns since the DNA had replicated.


7.1.9 Tandem repeats are used in DNA profiling.

A variable number tandem repeat (VNTR) is a short sequence of nucleotides that can be used to create a profile of a person based on how many times the sequence is repeated. The VNTR is found at the same locus (location on the chromosome) among different people making it relatively simple to find. DNA profiling can be used to solve criminal cases and solve parental disputes among other things


7.1.10 Analysis of results of the Hershey and Chase experiment providing evidence that DNA is the genetic material.

Hershey and Chase conducted an experiment to see whether proteins or DNA were the genetic material of the cell. To do this, they infected an E.coli bacteria with the T2 Phage virus. Viruses consist of only DNA inside a protein coat so there were no other variables in the mix.

DNA contains phosphorus but not sulphur, and proteins can contain sulphur but not phosphorous. This distinction was used in the experiment using isotopes (different forms) of sulphur and phosphorous. They made two strains of the T2 Phage bacteria, one with a heavier phosphorus isotope in the DNA, and one with a heavier sulphur isotope in the proteins. They did this by putting the T2 phage in a solution with everything necessary to replicate and only the heavier version of the sulphur or the heavier version of the phosphorous. After the T2 phage replicated a couple of times it would mostly consist of the heavier isotope

Hershey and Chase caused the T2 phage virus to inject its genetic material into the E. Coli bacteria. And then they put that E. Coli in a test tube and put that test tube in a centrifuge to spin it really fast in a circle. Because of the quick, circular motion the heavier parts of the E. Coli moved to the bottom of the test tube while the lighter isotopes were at the top.

When the sulphur strain injected its genetic material the percentage of heavy isotopes vs light isotopes were negligible. However, when the phosphorous strain of the virus injected its genetic material into the bacteria 65% of the DNA in the E. Coli was of the heavy isotope based on the centrifuge results.


Step 2: Primer Binding

The leading strand is the simplest to replicate. Once the DNA strands have been separated, a short piece of RNA called a primer binds to the 3' end of the strand. The primer always binds as the starting point for replication. Primers are generated by the enzyme DNA primase .

Enzymes known as DNA polymerases are responsible creating the new strand by a process called elongation. There are five different known types of DNA polymerases in bacteria and human cells . In bacteria such as E. coli, polymerase III is the main replication enzyme, while polymerase I, II, IV and V are responsible for error checking and repair. DNA polymerase III binds to the strand at the site of the primer and begins adding new base pairs complementary to the strand during replication. In eukaryotic cells, polymerases alpha, delta, and epsilon are the primary polymerases involved in DNA replication. Because replication proceeds in the 5' to 3' direction on the leading strand, the newly formed strand is continuous. The lagging strand begins replication by binding with multiple primers. Each primer is only several bases apart. DNA polymerase then adds pieces of DNA, called Okazaki fragments , to the strand between primers. This process of replication is discontinuous as the newly created fragments are disjointed.

Once both the continuous and discontinuous strands are formed, an enzyme called exonuclease removes all RNA primers from the original strands. These primers are then replaced with appropriate bases. Another exonuclease “proofreads” the newly formed DNA to check, remove and replace any errors. Another enzyme called DNA ligase joins Okazaki fragments together forming a single unified strand. The ends of the linear DNA present a problem as DNA polymerase can only add nucleotides in the 5′ to 3′ direction. The ends of the parent strands consist of repeated DNA sequences called telomeres. Telomeres act as protective caps at the end of chromosomes to prevent nearby chromosomes from fusing. A special type of DNA polymerase enzyme called telomerase catalyzes the synthesis of telomere sequences at the ends of the DNA. Once completed, the parent strand and its complementary DNA strand coils into the familiar double helix shape. In the end, replication produces two DNA molecules , each with one strand from the parent molecule and one new strand.


7.00x Introduction to Biology and 7.05x Biochemistry or similar (biochemistry, molecular biology, and genetics).

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About this course

You’re acquainted with your DNA, but did you know that your cells synthesize enough DNA during your lifetime to stretch a lightyear in length? How does the cellular machinery accomplish such a feat without making more mistakes than you can survive? Why isn’t the incidence of cancer even higher than it is? And, if the DNA in each and every cell is two meters long, how is this genetic material compacted to fit inside the cell nucleus without becoming a tangled mess?

Are you ready to go beyond the “what" of scientific information presented in textbooks and explore how scientists deduce the details of these molecular models?

Take a behind-the-scenes look at modern molecular genetics, from the classic experimental events that identified the proteins involved in DNA replication and repair to cutting-edge assays that apply the power of genome sequencing. Do you feel confident in your ability to design molecular biology experiments and interpret data from them? We've designed the problems in this course to build your experimental design and data analysis skills.

Let’s explore the limits of our current knowledge about the replication machinery and pathways that protect the fidelity of DNA synthesis. If you are up for the challenge, join us in 7.28x Part 1: DNA Replication and Repair.

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Watch the video: Molekulargenetik 3 DNA-Replikation Ablauf in Zellen (January 2022).