Semi-conservative replication of Chromosomes in eukaryotes:

  • Autoradiography experiment in Vicia faba, by J.H. Taylor and his co-workers for the study of duplicating chromosomes in the root tip cells were first published in 1957. 
  • They reported that DNA in all the organisms has the inherent capacity of self-replication. 
  • The mechanism of DNA replication is so precise that all the cells derived from a zygote contain exactly similar DNA both in terms of quality and quantity. 
  • The replication takes place in interphase after every cell division.

Theoretically, there may be following three possible modes of DNA replication:

  1. Dispersive Method
  2. Conservative Method
  3. Semiconservative Method

Semiconservative mode is the most accepted of all.

DNA Replication is Semi-Conservative:

  • Watson and Crick model suggested that DNA replication is semi-conservative. 
  • It implies that half of the DNA is conserved. 
  • Only one new strand is synthesized, the other strand is the original DNA strand (template) that is retained. 
  • Each parental DNA strand serves as a template for one new complementary strand.
  • The new strand is hydrogen bonded to its parental template strand and forms a double helix. 
  • Each of these strands of the double helix contains one original parental strand and one newly formed strand.

Meselson and Stahl Experiment:

  • Mathew Meselson and Franklin Stahl proved experimentally that parental strands of a helix are distributed equally between the two daughter molecules. 
  • They made use of the heavy isotope 15N as a tag to differentially label the parental strands. E. coli was grown in a medium containing 15N labeled NH4Cl.
  • In this way both strands of DNA molecules were labeled with radioactive heavy isotope 15N in their purines and pyrimidines. 
  • Therefore both strands were heavy or H DNA. 
  • The bacteria were then transferred into a medium containing the common non-radioactive nitrogen 14N, which is a light medium. 
  • It was found that after one cell division daughter molecules had one 15N strand the other 14N strand. 
  • So this is a hybrid molecule, a heavy light of HL.

Fig. Semiconservative replication.

Enzymes Involved In DNA Replication:

Both the prokaryotic and eukaryotic cells contain three types of nuclear enzymes that are essential for DNA replication. These enzymes are nucleases, polymerases and ligases.

(i) Nucleases:

  • The polynucleotide is held together by phosphodiester bonds. 
  • The nucleases hydrolyse the polynucleotide chain into the nucleotides. 
  • It attacks either at 3′ OH end or 5′ phosphate end of the chain. 
  • The nucleases are of two types.

(a) Exonucleases:

  • The nuclease that attacks the outer free end of the polynucleotide chain is called exonuclease. 
  • It breaks phosphodiester bonds either in direction 5’→ 3′ or in 3’→ 5′ direction. 
  • The enzyme moves in either cases stepwise along the chain and removes nucleotides one by one. 
  • Thus, the whole chain is digested.

(b) Endonucleases:

  •  The endonucleases attack within the inner portion of one or the double strands. 
  • A nick is made on a double stranded DNA molecule. 
  • However, if the polypeptide chain is single stranded (e.g. in DNA viruses), the attack of endonuclease will render the chain into two pieces.
  • On double stranded DNA the nick contains two free ends that in turn act as template for DNA replication. 
  • Apart from this, the nicked double helix is distorted due to rotation of free molecules around its intact strand.

(ii) DNA Polymerases:

  • DNA polymerases carry out the process of polymerization of nucleotides and formation of polynucleotide chains. 
  • This enzyme is called replicase when it replicates the DNA molecules and is inherited by daughter cells. 
  •  In prokaryotes, three types of DNA polymerases e.g. 
  • polymerase I (Poly-I), 
  • polymerase II(Pol II), and 
  • DNA polymerase III (Pol III) are found, 
  • whereas in Eukaryotic cells contain five DNA polymerases: α, β, γ, δ, and ε. Polymerase γ is located in mitochondria and is responsible for replication of mitochondrial DNA.
  • The other four enzymes are located in the nucleus and are therefore candidates for involvement in nuclear DNA replication.
  • Polymerase β is most similar to E. coli DNA Pol I because its main function is associated with DNA repair, rather than replication. DNA polymerase β is mainly used in base-excision repair and nucleotide-excision repair.
  • DNA polymerase 𝝳 is the main enzyme for replication in eukaryotes. It also has 3’→5′ exonuclease activity for proofreading. DNA polymerase 𝜶’is main function is to synthesize primers.

(a) Polymerase I (Pol I):

  •  It is discovered by The Kornberg 
  • It is also known as Kornberg polymerase . 
  • It is a single peptide chain with a molecular weight of 109,000 D. 
  • One atom of zinc (Zn) per chain is present, therefore, it is metalloenzyme. 
  • In E. coli, approximately 400 molecules of Pol I are present.
  •  It is roughly spherical of about 65 A° diameters.

Pol I possesses several attachment sites 

such as:

(i) A template site for attachment to the DNA template,

(ii) A primer site of about 100 nucleotides contemporary to a segment of RNA on which the growth of newly synthesized DNA occur,

(iii) A primer terminus site containing a terminal 3’OH group at the tip, and

(iv) A triphosphate site for matching the incoming nucleoside triphosphates according to complementary nucleotide of DNA template.


In E.coli the following three types of functions of Pol I have been found.


  • it cannot synthesize a long chain. 
  • It synthesizes only a small segment of DNA in 5′ → 3′ direction
  •  it takes part in repair synthesis. 
  • In E.coli Pol I polymerize the nucleotides at the rate of 1,000 nucleotides per minute at 37°C.

Exonuclease activity:

3’ → 5′ exonuclease activity:

  • Pol I catalyses the breaking of one or two DNA strands in 3’ → 5′ direction into the nucleotide components 
  • Therefore, it is called 3′ → 5′ exonuclease activity. 
  • Pol I correct the errors made during the polymerization, and edits the mismatching nucleotides at the primer terminus before the start of strand synthesis. 
  • Therefore, the function of Pol I is termed as repair synthesis.

5′ → 3′ exonuclease activity:

  • Pol I also breaks the polynucleotide chain in 5′ → 3′ direction with the removal of nucleotide residues. 
  • Upon exposure of DNA to the ultraviolet light two adjacent pyrimidines such as thymines are covalently linked forming pyrimidine dimers. 
  • These dimers block the replication of DNA. 
  • Therefore, removal of pyrimidine dimers e.g. thymine dimers (T=T) is necessary.
  • Through 5′ → 3′ exonuclease activity, Pol I removes pyrimidine dimers. 
  • Secondly, DNA synthesis occurs on RNA primer in the form Okazaki fragments. 
  • Through 5′ → 3′ exonuclease activity Pol I remove RNA primer and seal the gap with deoxyribonucleotides.

(b) Polymerase II (Pol II):

  •  Pol II is a single polypeptide chain (MW 90,000) that shows polymerization in 5’ → 3’ direction of a complementary chain.
  • It also shows exonuclease activity in 3’ → 5’ direction but not in 5’ → 3’ direction. 
  • The polymerization activity of Pol II is much less than Pol I in E.coli cells.
  •  About 50 nucleotides per minute are synthesized. 
  • E.coli cells contain about 40 Pol II molecules.
  • The 3′ → 5′ exonuclease activity of Pol II shows that it also plays a role in repair synthesis or DNA damaged by U.V. light just like Pol I. 
  • In the absence of Pol I, it can elongate the Okazaki fragments. 
  • Therefore, Pol II is an alternative to Pol I.

(c) Polymerase III (Pol III):

  • DNA polymerase III is several times more active than Pol I and Pol II enzymes. 
  • It is the dimer of two polypeptide chains with molecular weight 1,40,000 and 40,000 D respectively. 
  • Pol III is the main po­lymerization enzyme that can polymerize about 15,000 nucleotides per minutes in E. coll.
  • Synthesis of a long template also occurs when an auxiliary protein DNA (co-polymerase II) is linked with Pol III and produced Pol III-co Pol II complex. 
  • In addition Pol III also shows 3’→ 5′ exonuclease activity like Pol II.
  • The 5’→ 3′ exonuclease activity is absent. 

(iii) DNA Ligases:

  • The DNA ligases seal single strand nicks in DNA which has 5’→ 3′ ter­mini. 
  • It catalyses the formation of phosphodiester bonds between 3′-OH and 5′-PO4 group of a nick, and turns into an intact DNA. 
  • There are two types of DNA ligases: E. coli DNA ligase and T4 DNA ligase. 
  • The E. coli DNA ligase requires nicotina­mide adenine dinucleotide (NAD+) as cofactor, 
  • whereas T4 DNA ligase uses ATP as cofactor for joining reaction of the nick (Fig 5.19).

DNA Helicase enzyme

  • This is the enzyme that is involved in unwinding the double-helical structure of DNA allowing DNA replication to commence.
  • It uses energy that is released during ATP hydrolysis, to break the hydrogen bond between the DNA bases and separate the strands.
  • This forms two replication forks on each separated strand opening up in opposite directions.
  • At each replication fork, the parental DNA strand must unwind exposing new sections of single-stranded templates.
  • The helicase enzyme accurately unwinds the strands while maintaining the topography on the DNA molecule.

SSB proteins: 

  • Bind to the single strands of unwound DNA to prevent reformation of the DNA helix during replication.

DNA primase enzyme

  • This is a type of RNA polymerase enzyme that is used to synthesize or generate RNA primers, which are short RNA molecules that act as templates for the initiation of DNA replication.


  • This is the enzyme that solves the problem of the topological stress caused during unwinding.
  • They cut one or both strands of the DNA allowing the strand to move around each other to release tension before it rejoins the ends.
  • And therefore, the enzyme catalysts the reversible breakage it causes by joining the broken strands.
  • Topoisomerase is also known as DNA gyrase in E. coli.
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