What is DNA replication?
In molecular biology, DNA replication is the biological process of producing two identical DNA copies. It is read by DNA polymerase in the 3′ to 5′ direction, meaning the nascent strand is synthesized in the 5′ to 3′ direction.
DNA replication is the biological process, in which cells create two identical copies of DNA from the original DNA molecule with high accuracy (fidelity rate).
Before the structure of DNA was known scientist wondered how organism creates faithful copies of themselves. The 1940s brought the revelation to the scientist that prove the DNA is the genetic molecule. But not until James Watson and Francis Crick conclude its structure. In which DNA could act as a templet for the replication and transmission of genetic information to become clear.
The basic mechanisms of DNA replication are similar across organisms. In this article, we will focus on DNA replication as it occurs in E. coli, But the mechanisms of replication are similar in humans and other eukaryotes.
Let’s take a look at the proteins and enzymes that follow up replication, and how they work together to ensure accurate and complete replication of DNA.
The Basic Idea of Replication
DNA replication is a semiconservative process. That means each strand of DNA molecule acts as a template for the synthesis of a new, complementary strand.
This process takes one starting DNA molecule to two “daughter” molecules. With each new DNA double helix containing one new and one old strand (parent strand). As we show in the figure, it looks like a very simple process and nothing interesting in it. But what’s most interesting about this process is how it’s executed by the cell.
Steps of DNA Replication
There are three main steps to DNA replication: initiation, elongation, and termination. In order to fit within a cell’s nucleus, DNA is packed into tightly coiled structures called chromatin, which loosens prior to replication, allowing the cell replication machinery to access the DNA strands.
1) Initiation of DNA Replication
During initiation, proteins bind to the origin of replication while helicase unwinds the DNA helix and two replication forks are formed at the origin of replication. During elongation, a primer sequence is added with complementary RNA nucleotides, which are then replaced by DNA nucleotides.
This is the step where DNA replication starts. But the main question is that is there any specific binding site to start replication or it starts at a random site?
Yes, there is a specific site where the replication of chromosomal DNA begins which is known as the origin of replication.
Replication fork or evets at the replication fork
Synthesis of DNA replication occurs at the replication fork. The place at which the DNA helix is unwound and individual strands are replicate. In the E. Coli cell origin of replication is the oriC site. This site consists of a 254 bp long DNA sequence.
The bacterial initiator protein responsible for triggering DNA replication is DnaA. it binds regions in oriC site throughout the cell cycle. But to initiate the replication, DnaA protein must bind with few particular oriC sequences which possess five repeats of 9 bp sequence (R site).
When DnaA binds with the oriC site after that it recruits a helicase enzyme also known as DnaB helicase. Helicases are responsible for separating (unwinding) the DNA strands just ahead of the replication fork, using energy from ATP hydrolysis. Loading of the DnaB helicase is the key stapes in the replication initiation. As helicase migrates toward 5’ to 3’ direction unwinding of DNA strands travels with it.
But there are two questions arise.
I) How singal stranded DNA seprated from each other?
DNA is highly unstable in the form of a single strand. So what cells can do to separate this single-stranded DNA from each other? In this case, cells have specific protein which is single-stranded DNA binding proteins (SSBs). this protein bind with DNA after Helicase unwinds the strand and keeps them separated.
II) How the remaining DNA double helix is deal with increased tension due to unwinding?
When helicase rapidly unwinds the double helix. It increases tension for the remaining DNA molecule. For reviling this tension topoisomerases take place in the DNA replication. The replication fork may rotate as 75 to 100 revolutions per second. This is important because rapid unwinding can lead to the formation of positive supercoils in the helix ahead of the replication fork. Topoisomerase changes the structure of DNA by transiently breaking one or two strands without altering the nucleotide sequence of the DNA.
2) Elongation of DNA Replication
During elongation, an enzyme called DNA polymerase adds DNA nucleotides to the 3′ end of the newly synthesized polynucleotide strand. The template strand specifies which of the four DNA nucleotides (A, T, C, or G) is added at each position along the new chain.
In this particular phase of replication execute two different but related operations (Or you can say that related processes also). That is the leading strand synthesis and lagging strand synthesis.
As we show above at the replication fork, several enzymes are important to the synthesis of both strands. Let’s take a short summary of those enzymes.
First of all, parent DNA is first unwound by DNA helicase enzyme, and the resulting topological stress is release by topoisomerases. Then each separate DNA strand is then stabilized by SSB proteins. Up to this point, the mechanism of DNA synthesis is similar for both strands. But from this point, the synthesis of leading and lagging strands is sharply different.
Before we jump on to the synthesis of leading and lagging strands. Let’s discuss why this strand is known as leading and lagging strands. Their name shows their role in replication,
Leading strand that means this strand leads the DNA synthesis. The lagging strand is synthesized slowly in comparison to the leading strand due to that it lags behind the other strand that’s why it called lagging strand.
Leading strand synthesis.
The synthesis of each strand begins with the synthesis of DNA dependent RNA primer (10 to 60 nucleotide sequence). Which is synthesize by DNA primase (DnaG protein). Next primase interacts with DnaB helicase and follows up the first RNA primer synthesis.
This primer synthesized in the opposite direction to the DNA helicase is moving. In effect, the 3’ to 5’ strand of DNA becomes the prime leading strand for DNA replication. Now, Deoxyribonucleotides are adding into the prime leading strand by a DNA polymerase III complex. which links to the DnaB helicase tether(bind) to the opposite DNA strand to restrict the movement of the DNA polymerase III.
Leading strand synthesis now proceeds continuously. Keeping pace (motion) with the unwinding of DNA at the replication fork.
Lagging strand synthesis:
DNA polymerase always proceeds in 5’ to 3’ direction. So how can both strands be synthesized simultaneously?
If both strands were synthesized continuously while the replication fork move. So one strand would have to undergo ‘3 to 5’ synthesis. This problem was resolved by Reiji Okazaki and colleagues in the 1960s.
Okazaki found that one of the new DNA strands is synthesized in short pieces, now these pieces of fragments are known as Okazaki fragments. This work ultimately led to the conclusion that one strand is synthesized continuously and others discontinuously.
Mechanism of Okazaki fragment formation
As we show RNA primer is synthesized by primase. And next DNA polymerase III binds to the RNA primase and adds deoxyribonucleotide (dNTPs).
In this level, the synthesis of each Okazaki fragment seems straightforward. But the reality is quite complex. Because DNA polymerase moves along with the 5’ to 3’ direction. So the one problem faced by all DNA replication machines is How to simultaneously and co-ordinately replicate two antiparallel DNA strands?
To deal with this problem DNA polymerase III uses the one set of its core subunits (the core polymerase) to synthesize the leading strand continuously. While the other two sets of core subunit from one Okazaki fragment to the next on the looped leading strand.
In vitro, there are only two sets of core subunits with DNA polymerase III holoenzymes that can synthesis both the leading strand and lagging strand. However, the third set of core subunit increases the efficiency of lagging strand synthesis as well as the processivity of the overall replisome.
Now let’s look no how this set of core subunit work.
As shown in the figure, when DnaB helicase tie in front of DNA polymerase III. It starts unwinds the DNA at the replication fork as it travels along with the lagging strand template in the 5’ to 3’ direction. Primase occasionally associates with DnaB helicase and synthesizes a short RNA primer.
Now a new sliding clamp is then positioned at the primer by the clamp-loading complex of DNA polymerase III. When the synthesis of the Okazaki fragment has been completed.
Replication halts and the core subunits of DNA polymerase III dissociate from their sliding clamps (and form the complete Okazaki fragment) and associate with the new clamp. This initiates the synthesis of a new Okazaki fragment. Two sets of core subunits may be engaging in the synthesis of two different Okazaki fragments at the same time.
Once an Okazaki fragment has been completed, its RNA primers are removed by DNA polymerase I or RNase H1. And this space is replaced with DNA by the polymerase. Now, the remaining nick was seal by the DNA ligase.
How DNA ligase seal nick DNA?
DNA ligase catalyzes the formation of a phosphodiester bond between a 3’ hydroxyl at the end of one DNA strand and a 5’ phosphate at the end of another strand.
As you read above, that DNA replication occurs with a very high-fidelity rate. It incorporates the wrong nucleotide once per 104– 105 nucleotides polymerized. The accuracy of replication relies on the ability of replicative DNA polymerases to select the correct nucleotide for the polymerization reaction. And remove mistakenly incorporated nucleotide using their exonuclease activity.
3) Termination of DNA Replication
Termination of DNA replication occurs when two replication forks meet on the same stretch of DNA, during which the following events occur, although not necessarily in this order: forks converge until all intervening DNA is unwound; any remaining gaps are filled and ligated; catenanes are removed; and replication proteins are unloaded.
E. Coli genome replication carried out by pairs of the replication fork. That meets at the origins of replication and then move the opposite. DNA replication finishes when the two-replication fork of the circular E. Coli chromosome meets again at a termination site (ter).
This Ter sequence is arranged on the chromosome to create a trap that a replication fork can enter but can not leave. On the other hand, the Ter sequence function as binding sites for the protein called “Tus”. Which are acts as a terminus utilization substance.
Now the Tus-Ter complex can arrest a replication fork form only one direction. Because only one Tus- Ter complex function per replication cycle- the complex first encountered by anyone of the replication fork.
At that point opposite replication fork generally halt when they collide (clash or meet). So, when the first (arrested) fork meets the Tus-Ter complex other fork halts. Next, the final few hundred base pair of DNA between these large protein complexes are then replicate (this mechanism is unknown yet).
But the question is that how fork movement is stop?
There are two problem that must be solve by replisome.
- Formation of the interlinked chromosomes called catenanes.
- Dimerized chromosome.
Catenanes are produced when topoisomerase break and rejoins the DNA strand to ease supercoiling ahead of the replication fork. Now the separation of catenated circles in E. Coli requires topoisomerase IV (a type II Topoisomerase). Topoisomerase IV breaks both strands of one molecule. Pass the other molecule through the break, and then rejoin the strands. Then the separated chromosomes then divide into daughter cells at cell division.
when two chromosomes joined together and form a single chromosome twice as long. Dimerized chromosomes are results from DNA recombination that sometimes occurs between two daughter molecules during DNA replication. The terminal phase of replication of other circular chromosomes, including many of the DNA viruses that infect eukaryotic cells, is similar.
Now let’s move on our most awaited question
What is the importance of replication and why we have to study DNA replication?
Let’s first discus importance of replication.
have you ever think about how our diffrent body cell work diffrently?.
It’s because of DNA as we know the DNA is the genetic material of the cell. It having the necessary information for the cellular mechanism. That used in protein synthesis and protein is the most essential part of the cell. So this information needs to be pass in the next generation for maintaining their function.
This information is essential for life. It helps the cell to function normally. There are millions of cells that died every day in our bodies or in the ecosystem. If this essential information does not pass to the next generation so the function of our body organs is not performed normally. To deal with this problem, the parental cell generates copies of their DNA and pass this to new daughter cells. The parental cell gives a blueprint of there work.
why we have to study DNA Replication?
The most important thing which come into our mind. Why we have to study this?
As we know DNA replication is the most essential process in the cell, without this life doesn’t exist. With the study of DNA replication, we know how cell passes their information to the next generation. And the most important thing we develop several molecular techniques which are based on replication.
For example: – polymerize chain reaction (PCR). PCR is widely used by the scientist for multiplication of DNA. This complete process is based on the principle of DNA replication.
Based on the replication process, scientists developed artificial plasmid. That has a replication site in their sequences. So that they use proteins and enzymes from the cell and replicate itself with cell.
Now a days our lots of therapeutic activities are based on DNA replication.
Because of the study of the DNA replication process scientists found. That the process of DNA replication is similar in bacteria and humans. But the enzymes which are used in the replication process is different in some stage. For targeting specific protein or enzyme. We have to study this mechanism of DNA replication in different organisms. Because every organism has its own replication mechanism.
For example: in E. Coli there are five DNA polymerase enzymes and eukaryotic cells possess only three DNA polymerases. Based on this difference, they design a drug that is actively targeting the enzymes and protein. That is actively participating in the DNA replication of bacterial cells.’ click the link below where you get the complete list of drugs with there active target protein or enzymes.