LEC 9: DNA Replication I Flashcards
DNA Replication
Process of duplication of the entire genome prior to cell division. Biological significance:
– Extreme accuracy of DNA replication is necessary in order to preserve the integrity of the genome in successive generations
Basic Rules of DNA Replication
- Semi-conservative, Semi-discontinuous
- Bi-directional
- Starts at the ‘origin’ and synthesises in the 5’ -3’ direction
- RNA primers required
Leading Strand vs. Lagging Strand
Leading Strand: Simple addition of nucleotides along one strand, as expected
Lagging Strand: Other daughter strand is also synthesized 5’→3’ because that is only way that DNA can be assembled
- Compensate for this by feeding the DNA strand through the polymerase, and primers and make many short segments that are later joined (ligated) together
Semi-Discontinuous Replication
- Anti parallel strands replicated simultaneously
- Leading strand synthesis continuously in 5’ - 3’
- Lagging strand synthesis in fragments in 5’ - 3’
Prokaryotic DNApol
Responsible for the synthesis of DNA
Modes of Action of DNApol (Distributive vs. Processive)
Remember that the synthesis of DNA occurs 5’ to 3’
• Distributive = Adds a single base to the primer and then dissociates
• Processive = Adds a number of bases to the primer and then dissociates
Processivity of Different Enzymes
Processivity of different enzymes differs
• DNA Polymerase I: processivity of 15-20 bases
• DNA Polymerase III core: processivity of 15-20
• DNA Polymerase III holo: processivity of 1,000’s
• DNA Polymerase IV: Distributive
DNApol Functions
5’-3’ Polymerase: synthesizes new DNA strands
5’-3’ Exonuclease: degrades DNA or RNA strands, removes damaged DNA and RNA primers during replication
3’-5’ Exonuclease: removes mistakes made during replication
The DNAPol 3’-5’ Exonuclease Proofreading Reaction
If an incorrect base is added to the strand…
- Is unable to pair with the template, thus inhibiting further synthesis
- The base is excised by 3’-5’ Exonuclease, allowing synthesis to continue
Semi-Discontinuous Replication
At the replication fork both strands of DNA grow in the same direction
• This means that 1 strand grows in a 5’-3’ direction, whilst the other grows in an apparent 3’-5’ direction
• DNA polymerase can only synthesize in a 5’-3’ direction
Synthesis of the Lagging Strand
- The RNA primer is extended by DNA polymerase III until it reaches the 5’ end of the next primer
- DNA pol III then dissociates, and DNA polymerase I binds, replaces the RNA primer with DNA
- DNA ligase seals the gaps
Primer Removal using 5’-3’ Exonuclease
- DNA synthesis stops at the 3’ end of the preceding primer leaving a nick
- DNA polymerase 1 binds to the nick
- Degradation of the primer and synthesis of new DNA occurs simultaneously
- DNA ligase seals the nicks
E.Coli DNApols
Pol1: repair, primer removal, 5'-3' exonuclease Pol2: translesion synthesis Pol3: Replication Pol4: translesion synthesis Pol5: translesion synthesis
Eukaryotic DNApols
α: Lagging strand synthesis δ: Leading strand synthesis, repair β: Repair γ: Replication ε: Replication/repair
Translesion Synthesis
- Non-coding lesion in the DNA template causes inhibiiton of DNA synthesis
- Error prone polymerases insert random bases opposite lesion
