LSM1401 Summary 8 © Lim Fang Jeng
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DNA Replication
DNA replication is semi-conservative. (i.e. New DNA=Template strand+ New strand) (Meselson-Stahl experiment)
- Requirements of the process o Templates
o RNA primer o Free dNTPs
o Enzymes and proteins
- In vitro: DNA primer is used (more stable) - In vivo: RNA primer
- Synthesized from 5’3’ , but the polymerase read the template strand FROM 3’5’!!!!!!!!!!!!!!!
- Reason:
o Breaking of phosphate group has a lot of energy, it is easier to form the phosphodiester bond
Replication
- They are bidirectional - Two replication forks
o Leading strand
o Lagging strand (Okazaki’s fragment)
- Always synthesized from 5’3’
Prokaryotes
Initiation factors: DNA A, B, C & T
(1) Unwinding of double helix by helicase (2) Compensation of super coiling by DNA
gyrase
(3) Single strand is maintained by SSA (Single stranded binding proteins) Steps:
(1) After initiation, primase will synthesize a RNA primer
(2) DNA pol III will take over and synthesize the remaining nucleotides
(3) In lagging strand, DNA pol III will stop since there is a discontinuous synthesis (4) DNA pol I will come and remove the RNA primer AND replace it with DNA (5) DNA ligase seals the nicks, replication continues….
LSM1401 Summary 8 © Lim Fang Jeng
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Eukaryotes
Similar with prokaryotes, with different enzymes and proteins
- Topoisomerase (= Gyrase) relieves the torsional strain of the unwinding - Replication protein A (RPA)(=SSB) maintains the single stranded DNA - Helicase (T antigen) – Unwinds the DNA
- DNA pol α & DNA pol
- DNA pol α work with primase ( as such, it is very inefficient) (Polymerizing enzyme) - DNA pol will take over and finishes the replication (Main polymerizing enzyme) - Removal of RNA primer: RNAseH1 and FEN-1
- DNA ligase seals the nicks
Prokaryotes Eukaryotes
Polymerases:
I: synthesis, proofreading, repair, RNA primer removal II: Repair enzyme
III : Main polymerizing enzyme
IV, V: Repair enzymes under unusual conditions
Polymerases:
α: Polymerizing enzyme (Inefficient, works with primase)
β: Repair enzyme
: mitochondrial DNA synthesis : Main polymerizing enzyme : Unknown
DNA pol I has 5’3’ exonucleases activity
The rest are 3’5’ exonucleases No DNA pol has this function
DNA pol α and δ are 3’5’ exonucleases
One origin of replication Several origins
No histones Histones complexed to DNA
Okazaki fragments: 1000-2000 Okazaki fragment: 150-200
High Fidelity
- Before covalent bonding of nucleotides:
o Correct base pairing: correct nucleotides has a higher affinity for the moving polymerase than does the incorrect nucleotide
o Conformational change double-check before it catalyses covalent bonding of the nucleotide: INCORRECTLY BOUND NUCLEOTIDE ARE MORE LIKE TO DISSOCIATE
- After covalent bonding of nucleotides:
o 3’5’ proofreading exonuclease: clips off improperly-paired nucleotide at 3-OH end of primer strand
o Using E(Editing domain) to cut phosphodiester bond from P (Polymerizing) domain
LSM1401 Summary 8 © Lim Fang Jeng
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5’ 3’ Chain Growth
- When there are mistakes in the chain, if the DNA grows from 3’5’, it would be not enough energy to change the base pairs
- It allows the chain to be elongated when a mstake is being removed
Speed of Replication
- Replication is a rapid process - 50-100 nucleotides per second
- Bacteria is even faster (1000 nucleotides/s)
