Replication of DNA

Introduction to DNA replication

Replication of DNA is a semi-conservative process where the daughter's DNA retains the originality of the parental strands. DNA replication is not a simple process as there are 3 billion DNA double strands in the human genome that need to be replicated. Replications necessitates a lot of energy, enzymatic processes, and a significant number of catalysts. Although it is an automatic process, the daughter cells must receive the genetic material as exactly as that of parental strands. As an experimental proof for DNA replication, Matthew Meselson and Franklin Stahl conducted a trial on E-coli. They have chosen  E-coli as the  DNA replication sample because E-coli resembles the human DNA. In the year  1958, they have explained the process as follows.

 

Table of Contents

1. Introduction to DNA replication

2. Triggers and sources of DNA replication

3. DNA polymerase reaction

4. Transcription

5. Steps of transcription

6. Transcription unit

7. Mechanism of Transcription

      7. 1 Activation of Ribo-nucleotides

      7.2  DNA Templateformation

              7.2.1 Base Pairing

              7.2.2 Chain Formation

              7.2.3 Separation of RNA

              7.2.4 Duplex Formation

 

 

 

DNA -replication ( semi-conservative )

 

 

Triggers and sources of DNA replication

The replication of DNA is a semiconservative process where each parental DNA  of a double helix is an original template from which 2 new daughter strands erupt as shown in the image. The pair is known as a complementary strand. There are many enzymes supporting the whole process but the most primary supporting factor is the polymerases. Polymerases need an original template and a starter called primer. During the DNA replication, one new strand called the leading strand will be formed. Leading strand in a template becomes 2 small pieces (daughter cells) and these are called the lagging strands. The additional enzymes like DNA helicase, DNA primase, DNA ligase, and topoisomerase are also helping in DNA replication. The process is a semi-conservative because the replication process brings out two "daughter" molecules by a double-strand with each newly formed double helix containing one new daughter cell and one old parental strand( Half- saved from the original and the raining half is added as new daughter strand). This means, out of 2 pairs, 1 pair has 2 parents and a daughter and the other pair also has a parent and 1 daughter cell. In order for a parental DNA to replicate in a normal way, cells need to copy their DNA and pass them to the daughter's DNA with few errors or in an error-free manner. One of the key processes involved here is the DNA polymerase reaction.

 

DNA polymerase reaction

DNA Polymerase chain reaction

DNA Polymerase reaction

 

 

 

1. Replication always starts at specific locations on the DNA, which are called origins of replication and are recognized by their sequence

2. DNA polymerase is an enzyme that assists in synthesizing the DNA molecules from deoxyribonucleotides. Deoxyribonucleotides are the building blocks of DNA. These enzymes work in pairs to generate 2 identical DNA strands from an original parent template. During this process, DNA polymerase "reads" the existing DNA strands to create two new strands that match the existing ones. The formula is, Deoxynucleoside triphosphate + DNA ⇌diphosphate + DNAn+1. DNA polymerase will slowly add one by one of the nucleotides to the growing DNA chain. They only integrate the nucleotides which are complementary to the template.

3. DNA polymerase must be highly efficient because they have to catalyze the polymerisation of many nucleotides in a quick time.  One of the examples is, E-coli that has only 4.6 × 106 base pairs compared to the diploid content of human which is 6.6 × 109 bp. The process of polymerization hardly takes 38 minutes or in other words, the rate of polymerization is 2000 base pairs per second.

4. Another enzyme, deoxyribonucleoside triphosphate which act as substrates that provide the energy needed for the polymerisation reaction.

5. In addition to the enzymes, there another tool called replication fork (see image on the right) helps in separating the lengthy parental  DNA strand.

6. DNA ligase is another enzyme that assists in the replication of DNA. The DNA-dependent DNA polymerases catalyze polymerization only in one direction that is 5’ to 3’. This creates some additional complications at the replicating fork. Consequently,  on one strand  (the template with polarity 3' to 5,  the replication is continuous, while on the other (the template with polarity  5' to 3' ),  it is discontinuous.  The fragments that are synthesized in a disorderly fashion are later joined by the enzyme DNA ligase. It is because, the origin of replication that a piece of DNA is needed to be propagated during recombinant DNA procedures requires a vector. Hence vectors provide the origin of replication. Abnormal replication like a failure in cell division after DNA is being replicated leads to a condition called polyploidy (a chromosomal anomaly).​

 

Transcription

The process of copying genetic information from one strand of the DNA into RNA is termed as transcription. It is the initial step in gene expression where a segment of template DNA is copied into mRNA (messenger RNA) through the enzyme RNA polymerase. During the process of transcription, a sequence of DNA scanned by an RNA polymerase, which produces a complementary and antiparallel RNA strand called a Primary transcript.

 

Steps of transcription

1. RNA polymerase, together with one or more general transcription factors, binds to promoter DNA.

2. RNA polymerase creates a transcription bubble, which separates the two strands of the DNA helix. This is done by breaking the hydrogen bonds between complementary DNA nucleotides.

3. RNA polymerase adds RNA nucleotides (which are complementary to the nucleotides of one DNA strand).

4. RNA sugar-phosphate backbone forms with assistance from RNA polymerase to form an RNA strand.

5. Hydrogen bonds of the RNA–DNA helix break, freeing the newly synthesized RNA strand.

6. If the cell has a nucleus, the RNA may be further processed. This may include polyadenylation, capping, and slicing.

7. The RNA may remain in the nucleus or exit to the cytoplasm through the nuclear pore complex.

 

Transcription unit

A transcription unit is a sequence of nucleotides in DNA that codes alongside the single RNA molecule in a way that requires information in the sequence. A transcription unit normally has a promoter, an RNA-coding sequence, and the terminator. There is a narrow principle that transcription can occur only in 5’→ 3′ direction and such strand of DNA is called template strand/ master strand.

 

Mechanism of Transcription:

1. Activation of Ribo-nucleotides

In this process, Enzyme phosphorylase along with the energy, phosphorylated Ribonucleotides will get activated. The activated Ribonucleotides are adenosine triphosphate (ATP), guanosine triphosphate (GTP), uridine triphosphate (UTP) and cytidine triphosphate (CTP). This process is called phosphorylation.  Which happens prior to the  Transcription, the nucleotides are activated through phosphorylation.

 

2. DNA Template

A. In order to form a template, 3 major regions are required, namely, initiation site, a promoter region, coding region, and a terminator region. As the name indicates, the initiation site launches/starts the transcription.

B. Promoter region has an RNA polymerase recognition site and RNA polymerase binding site.

C. In a coding region, the chain opening occurs. Chain opening needs unwindases, gyrases, and single-stranded binding proteins.

D. In a terminator region, the two strands of DNA uncoil progressively from the site of polymerase binding. One of the two strands of DNA (3’—» 5′) functions as a template for transcription of RNA. It is called master, template or antisense strand.

3. DNA strand formation

1.Base PairingThe Ribonucleoside triphosphates come closer to lie just opposite the nitrogenous bases of the DNA template (Antisense strand). This results in the formation of complementary pairs, I,e  they form complementary pairs, U goes opposite to A, A goes opposite to T, С goes opposite to G, and G goes opposite to C. A pyrophosphate is released from each ribonucleoside triphosphate to form Ribonucleotides. The pyrophosphate is hydrolyzed with the help of enzyme pyrophosphatase. It releases energy.

2.Chain Formation: With the help of RNA polymerase the adjacent ribonucleotides are held tightly against the DNA template to form an RNA chain.

3.Separation of RNA: Separation is enhanced by the factor called ATPase activity. This helps in the release of the completed RNA chain from the previous phase of chain formation. The released RNA is called primary transcript which will further get processed to form a functional RNA.

4.Duplex Formation: Once the primary transcript is released, the 2 strands of DNA form linkages amongst complementary base pairs. At this stage, Gyrases, unwindases and SSB proteins are released, consequently, there is double-helical DNA formation.

 





img-1

img-1

img-1

Course List