DNA - An overview

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Slide 73

To prepare DNA for replication, many proteins are involved in replication

These proteins are required because DNA must be single-stranded before replication can proceed.

The following are important Protein and enzyme required for DNA replication:

1. DNA helicases

2. Single stranded DNA binding proteins (SSB)

3. Topoisomerases / DNA gyrase

4.Primase

5. DNA Polymerases 6. Sliding DNA clamps

7. RNAse H 8. DNA ligase

Slide 74

DNA Helicases - These proteins bind to the double stranded DNA and stimulate the separation of the two strands.

DNA single-stranded binding proteins - These proteins bind to the ssDNA as a tetramer and stabilize the single-stranded structure that is generated by the action of the helicases.

Their binding exhibits cooperativity (the binding of one tetramer stimulates the biding of additional tetramers)

Replication is 100 times faster when these proteins are attached to the single-stranded DNA.

Slide 75

DNA Gyrase - This enzyme catalyzes the formation of negative supercoils that is thought to aid with the unwinding process.

It catalyzes the removal of Positively supercoils in DNA, which considered to be essential for replication and are believed to play a key role in unwinding process .

Primase – DNA replication require RNA primers to begin.

Primase is a specialized RNA polymerase which make short RNA primers using ssDNA as a template

Primase activity requires the formation of complex of primase and at least six other proteins.

This complex is called Primosome

Slide 76

DNA Polymerase: The synthesis of DNA is catalyzed by DNA Polymerase.

It can add only dNTPs to the 3’ and form polynucleotide.

Sliding DNA Clamps: It is to increase the degree of processivity of the DNA Polymerase sliding DNA clamps surrounds the DNA and binds to the DNA polymerase and holding them together.

RNAse H: To complete the DNA replication, RNA primers must be removed.

RNAse H Specifically degrade RNA that base paired with DNA. (H stands for Hybrid as RNA – DNA Hybrid)

Slide 77

DNA Ligase - Nicks occur in the developing molecule because the RNA primer is removed and synthesis proceeds in a discontinuous manner on the lagging strand.

Slide 78