MOLECULAR GENETICS 8

 Machinery & Enzymes for Replication

Enzymes play an important role acting as catalysts during the process of DNA replication. Some of the important enzymes are:

• DNA polymerase
• Helicase
• Primase
• DNA ligase

Energy source is needed to provide energy during the replication process.

 Deoxyribonucleoside triphosphates act as substrates & provide energy for polymerization reaction.

DNA polymerase

DNA polymerase creates DNA from nucleotides.

Also known as DNA dependent DNA polymerase

 It reads the existing DNA strands to create two new strands that match the existing ones. 

This enzyme is needed every time a cell divides so that one copy of DNA can be passed to each daughter cell.

DNA polymerase is a highly efficient enzyme, as it can replicate a large number of base pairs in a very short time. 

Rate of replication or Rate of polymerization is approx 2000 bp per second. 

A total of 4.6 * 106 base pairs are replicated within 18 minutes.  

DNA polymerase also catalyze with high degree of accuracy. A mistake is made once in every 1 billion base pairs copied. 

DNA polymerase proof reads to check for errors. However, these errors if remain can cause mutations.

Helicase

Enzyme Helicase unwinds DNA from tightly double stranded structure.

 Only after the strands are separated, DNA polymerase can do its job of creating the new strands. 

This enzyme separates the strands by breaking the hydrogen bonds between the bases of the two strands.

Primase

This enzyme creates a short fragment of RNA (primer) paired with the template DNA strand. 

This enzyme initiates the process of creation of new strands. 

DNA polymerase cannot initiate the process on its own. Therefore, primase initiates the same

Ligases

It binds various small fragments of DNA.

Process of DNA replication

Replication cannot be initiated in any random part of DNA. 

Region in a DNA where replication initiates is termed as ‘Origin of Replication’. (Ori)

Prokaryotes- usually 1 ori

Eukaryotes- multiple

Step 1.

Enzyme Helicase breaks hydrogen bonds, thus separating the two strands of DNA. Replication fork structure is formed.

Step 2.

New strand formation- only in one direction 5' - 3'

1. Continuous synthesis takes place in the Leading strand. (3' -5') 
2.  In this strand, DNA is synthesized in the same direction as the growing replication fork. 

1. Discontinuous synthesis takes place in the Lagging strand. 
2. 
   
3. DNA polymerase can add new free nucleotides to the 3’ end of the new strand. In the lagging strand, no free 3’-OH end is available. 
4. Therefore, DNA polymerase is unable to initiate the process. 
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6. Enzyme Primase initiate the process by creating a small RNA fragment called Primer. 
7. 
   
8. DNA polymerase then extends the primed segments adding free nucleotides. 
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10. RNA primers are replaced with DNA.
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12.  Thus, we have DNA fragments. direction of synthesis in Lagging strand (It is opposite to the direction of growing replication fork). DNA Ligase now joins the DNA fragments and forms a complete DNA.

These DNA fragments are termed as ‘Okazaki fragments’ after the name of the scientist who first described the process of Discontinuous synthesis on Lagging strand.

Later, fragments are joined by Ligase enzyme.

This entire process of DNA replication occurs during S-phase of cell cycle in eukaryotes. Research is still going on for more detail on the replication process.