DNA Replication and Enzyme Activities

Study Notes

Post Replication Modifications

DNA polymerase III and I have a 3’ to 5’ exonuclease activity which is also known as proofreading. This ensures that the newly added nucleotide is correct and complementary to the template.
If an incorrect nucleotide is added, the enzyme removes it and replaces it with the correct one.

Differences between 5′→3′ and 3′→5′ exonucleases

The 5’ to 3’ exonuclease activity of DNA polymerase I removes one nucleotide at a time from an RNA primer that is properly base-paired to the template. This is necessary for the excision of RNA primers.
The 3’ to 5’ exonuclease activity can remove incorrect deoxynucleotides in the 3’ to 5’ direction. This is important for proofreading.

DNA Pol I

DNA Pol I has three activities:
- Polymerase activity from 5’ to 3’ for the synthesis of DNA segments replacing RNA primers.
- Exonuclease activity from 5’ to 3’ for excision of RNA primers and repair.
- Exonuclease activity from 3’ to 5’ for proofreading activity during the synthesis of DNA segments.

Eukaryotic DNA Replication

The process of eukaryotic DNA replication is similar to that of prokaryotic DNA synthesis, with some differences:
- Multiple origins of replication in eukaryotic cells versus single origins of replication in prokaryotes.
- In eukaryotes, RNA primers are removed by RNase H and FEN1 rather than by a DNA polymerase.

Eukaryotic DNA Polymerase

At least five key eukaryotic DNA polymerases have been identified and categorized based on:
- Molecular weight
- Cellular location
- Sensitivity to inhibitors
- The templates or substrates on which they act
They are designated by Greek letters.

Packing of Eukaryotic DNA

Eukaryotic DNA is packaged into chromosomes.

DNA Replication

The transmission of the genetic information found in parent DNA to daughter cells.
The synthesis of daughter DNA from parent DNA.
A process in which DNA copies itself to produce identical daughter molecules of DNA.

Character of Replication

DNA replication is semiconservative - A parent DNA has two strands complementary to each other. Both strands undergo simultaneous replication to produce two daughter molecules. Each newly synthesized DNA has one half of the parental DNA (one strand from original) and one half of new DNA.

Overview of DNA replication

Separation of the two complementary strands of the parental DNA.
Formation of the replication fork.
Formation of an RNA primer.
Chain elongation – a new strand is formed by base pairing, complementary with the parent strand.
Excision of RNA primers and their replacement by DNA.
Two molecules are made, each with one new and one old DNA strand.

Steps of DNA Replication in Prokaryotes

Separation of the parental 2 DNA strands (unwinding)
- Initiation of DNA synthesis occurs at a site called the origin of replication.
  - There is a single site in prokaryotes, whereas in eukaryotes, there are multiple sites of origin.
  - These sites mostly consist of a short sequence of A-T base pairs.
- A specific protein called dnaA binds with the site of origin for replication.
- The two complementary strands of DNA separate at the site of replication by DNA helicase to form a bubble.
- Multiple replication bubbles are formed in eukaryotic DNA molecules, which is essential for a rapid replication process.
- DNA helicases separate the double helix by cleaving the hydrogen bonds between the two DNA strands.
- Single strand DNA binding proteins (SSBP) prevent the two DNA strands from rejoining and protect the template from nucleases that cleave single-stranded DNA.
- As the two strands are separated, this creates coils in front of the separated part (supercoils) which prevents further separation of the helix.
- DNA topoisomerases remove supercoils.
Function of topoisomerases
- Topoisomerases have both nuclease (strand cutting) and ligase (strand resealing) activities.
- They make a transient cut (in the phosphodiester bond) in one strand (topoisomerase I) or both strands (topoisomerase II).
- This cut relaxes the supercoils and then topoisomerase reseals the cut (reforms the phosphodiester bond).
- DNA gyrase, a type II topoisomerase, is found in bacteria and plants.
Direction of DNA replication:
- DNA replication is bidirectional, meaning that the synthesis of two new DNA strands, simultaneously, takes place in the opposite direction.
  - One is in a direction (5’→3’) towards the replication fork, which is continuous, the other in a direction (5’→3’) away from the replication fork, which is discontinuous.
Synthesis of new DNA strands by DNA polymerase enzyme:
- Notes on DNA polymerases:
  - They cannot initiate DNA synthesis; they can only add a new complementary deoxynucleotide to a preexisting nucleic acid chain (RNA primer).
  - They can only synthesize DNA in one direction from 5’ to 3’ (as they are able to read the DNA template in one direction 3’ to 5’).