Question

Use the six enzymes used during DNA replication to explain the process of DNA replication. Include the function and purpose

Answer 1

Six enzymes involved in DNA replication are

i) Helicase

ii) Gyrase

iii) Primase

iv) DNA polymerase III

v) DNA polymerase I

vi) Ligase

Steps involved in DNA replication:

i) DNA helicase cannot open up an intact ds DNA strand so it needs a partially unwound region in DNA to start the replication. There is two types of AT rich sequences in Ori C region of ds DNA, 9 mer and 13 mer repeats. Dna A protein binds to the 9 mer repeats and form an initial complex. This complex creates tortional stress due to the over winding of the DNA at the 9 mer region. The tortional stress has to be compensated by underwinding in neighboring 13 bp region and it leads to melting of duplex DNA at the 13 mer region of ori C.

ii) Now Dna B or helicase (a hexamer) is loaded onto the partially open up region in association with the helicase loader Dna C (also a hexamer) in an ATP dependent manner. Further unwinding by helicase requires ATP hydrolysis which extends the replication fork. The intrastrand base pairing in ss DNA region is prevented by single stranded DNA binding (SSB) protein.

iii) Unwinding of ds DNA strands by helicase creates a torque in the duplex which is removed the the enzyme gyrase or type II topoisomerase.  

iv) DNA polymerase can't perform de-novo synthesis because they are primer dependent. Therefore cell uses a special RNA polymerase called primase to synthesis a short 10-12 bp RNA primers. Primase is activated by helicase. Once the SSB proteins are removed by Pri A protein, Primase can synthesize the primers.

v) Finally the main enzyme of DNA synthesis DNA polymerase III binds to the primer template junction in association with clamp loader and starts DNA polymerization. Two DNA polymerase work at the replication fork at the same time, one for leading strand and one for lagging strand synthesis.

vi) Once each okazaki fragment formation completes, DNA polymerase I removes the RNA primer by its 5'-3' exonuclease activity.

vii) Finally the gaps created by removal of RNA primers are joined by DNA Ligase. It catalyses the phosphodiester bond formation between 3' OH of of one end of DNA fragment and 5' Phosphate group of the next DNA fragment.