The machinery and the Enzymes / DNA

Replication Mechanism

• The process of replication in living cells requires a set of enzymes.
• THE main enzyme is referred to as DNA-dependent DNA polymerase.
• It has the ability to polymerize some 2000 bp per sec.
• Any mistake during replication would result into mutation.

DNA replication completes in following steps :-
i) Origin of replication
ii) Unwinding of helix

Origin of replication

• Replication begins at a particular region of DNA which is called origin of replication. It is called Ori-C in E.coli.
• Prokaryotes have single origin of replication, On the other hand eukaryotes have several thousands origin of replication.

Unwinding of helix

It is brought about by enzymes helicase, which is ATP dependent.
Replication fork – Unwinding of DNA molecule into strands results in the formation of Y-Shaped structure called replication fork.

Formation of Strands

Enzyme known as Primase synthesize a short primer strand of RNA.

DNA replication would not occur without enzymes that catalyze various steps in the process. Enzymes that participate in the eukaryotic DNA replication process include:

DNA helicase – Unwinds and separates double stranded DNA as it moves along the DNA. It forms the replication fork by breaking hydrogen bonds between nucleotide pairs in DNA.
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Primase – Primase initiates the process by creating small RNA segments called RNA primer.
DNA polymerases – it extends the primed segments by adding free nucleotides.
Topoisomerase – Unwinds and rewinds DNA strands to prevent the DNA from becoming tangled or supercoiled.
DNA ligase – Joins DNA fragments together by forming phosphodiester bonds between nucleotides.

Elongation of New Strand

Continous synthesis (leading strand)

• DNA polymerase catalyses polymerization only in the direction. That is 5′ 3′.
• This creates some additional complication at the replication fork. Consequently the replication is continuous on One template strand with polarity 3’5′. It is known as leading daughter strand.

Discontinous synthesis (lagging strand)

• The replication is discontinuous in the form of okazaki fragments on the other template strand with polarity 53′ . It is known as I daughter strand.
• Primase initiates the process by creating small RNA segments called RNA primer.DNA polymerase extends the primed segments by adding free nucleotides. RNA primers are removed and replaced with DNA.
• The discontinuously synthesized fragments are later joined by enzyme called DNA ligase.