DNA Replication Overview and Stages

DNA replication is the process of making a copy of a DNA molecule
This process is essential for cell division and maintaining genetic information
The process occurs in a semi-conservative manner, meaning each new DNA molecule consists of one original strand and one new strand

Template: The original DNA molecule serves as a template
Enzymes: Various enzymes are needed for the process, including DNA polymerase, helicase, primase, and ligase
Nucleotides: Free nucleotides (dATP, dGTP, dCTP, and dTTP) are needed as building blocks for the new strand
A starting point: An origin of replication is necessary to initiate the process

Initiation: The DNA molecule unwinds, forming replication forks, where the new strands of DNA will be built.
Elongation: DNA polymerase adds nucleotides to the 3' end of the existing strand, following the template strand
- Leading strand: Synthesized continuously in the 5' to 3' direction
- Lagging strand: Synthesized discontinuously in short fragments (Okazaki fragments), also in a 5' to 3' direction
- DNA polymerase synthesizes both strands
- Primer required: A short RNA primer is needed for elongation to start
  - Primase - makes the primer
  - DNA polymerase III starts at the primer
Termination: Replication ends when the two replication forks meet and the replication process is complete, creating two identical DNA molecules. The two molecules are separated and the process is complete.
Multiple enzymes and proteins: Enormous amounts of enzymes and proteins are needed for each strand

Similarities: Both use a semi-conservative process of DNA replication requiring a template, building blocks, enzymes, and a starting point
Differences: Eukaryotes have many origins of replication, while prokaryotes have only one origin. Eukaryotic replication takes longer than prokaryotic replication

Single circular chromosome vs Multiple linear chromosomes
Replicons and Origins
Bidirectional synthesis: Replication proceeds in both directions from the origin. Creates two replication forks moving outwards
Enzymes:
- Helicase: Unwinds the DNA double helix
- Primase: Synthesizes RNA primers
- DNA Polymerase III: Adds nucleotides to the growing DNA strand
- DNA Polymerase I: Removes RNA primers and replaces them with DNA
- DNA Ligase: Joins the Okazaki fragments of the lagging strand

Specific DNA sequence: Origin of replication (OriC) - starting point in prokaryotes
DnaA binds OriC to start unwinding the DNA strands
Helicase unwinds the DNA molecule into two strands. This creates a replication fork
Single strand binding proteins (SSBs) prevent DNA from re-annealing
Topoisomerase reduces the strain placed on DNA ahead of the replication fork during unwinding of the DNA
Primase creates short complementary RNA sequences to the DNA strands (Primers)
Primase begins DNA replication process for leading strand
This occurs because DNA polymerase require a 3' OH group to add new nucleotides

Leading strand: DNA polymerase can add nucleotides continuously to the 3' end of the leading strand
Lagging strand: Synthesized in small fragments (Okazaki fragments)
- DNA polymerase requires a primer that is complementary to the DNA strand being copied
- Fragments are joined together by the enzyme DNA ligase

DNA polymerase III adds nucleotides to the 3' end of the growing DNA strand in a 5' to 3' direction, the new strand follows the existing strand
Nucleotides: The template strand determines which nucleotides are added to the growing strand by base pairing rules
- A with T; C with G
Directionality: DNA polymerase adds nucleotides only to the 3' end of the growing strand, therefore new DNA strands are synthesized 5' to 3'
- Leading strand elongation: Continuous
- Lagging strand elongation: Discontinuous – new fragments (Okazaki fragments)
Importance of proofreading: Involves 3'-to-5' exonuclease activity of DNA polymerase – crucial for accuracy

short, newly synthesized DNA fragments that are formed on the lagging template strand during DNA replication
Fragments are joined together by DNA ligase to create a continuous lagging strand

Replication ends when the two replication forks meet and the DNA molecule is replicated into two single stranded molecules

There are many polymerases and they have various functions, but the most important for DNA replication is DNA polymerase III
Proofreading: 3' to 5' exonuclease activity - checks for errors before adding new nucleotides.
Other processes in DNA replication:
- Mismatch repair to correct errors
- Environmental damage repair mechanisms
Mutations: DNA replication errors can lead to mutations, alterations of the DNA sequence, that is sometimes essential to evolution

More complex than prokaryotic replication
Multiple origins of replication (versus one in prokaryotes)
- Initiation sites
- Many origins allow for faster replication of larger genomes
Nucleosomes need to be dealt with

Specialized structures at the ends of eukaryotic chromosomes
Protect from degradation
Repeat sequences
Telomerase (an enzyme): uses an RNA template to synthesize telomere sequences, maintains the integrity of telomeres

DNA polymerases have proofreading mechanisms (3'-5') to correct errors during DNA replication
Mismatch repair: Corrects errors missed during replication
- Enzymes scan the newly replicated DNA for mismatches
- Enzymes cut out the mismatched DNA section
- DNA polymerase fills in the gap, DNA ligase seals