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DNA REPLICATION
Anna Onofri
Liceo «I.Versari»
Learning objectives
1. Understand the basic rules governing DNA replication
2. Understand the function of key proteins involved in a generalised replication model
“It has not escaped our notice that the specific pairing we
have postulated immediately suggests a possible copying
mechanism for the genetic material”
Watson & Crick
Nature (1953)
Original drawing by Francis Crick
Four requirements for DNA to be genetic material
Must carry information• Cracking the genetic code
Must replicate• DNA replication
Must allow for information to change• Mutation
Must govern the expression of the phenotype• Gene function
Much of DNA’s sequence-specific information is
accessible only when the double helix is unwound
Proteins read the DNA sequence of nucleotides as the
DNA helix unwinds.
Proteins can either bind to a DNA sequence, or initiate
the copying of it.
DNA stores information in the sequence of its bases
Some proteins recognize the base
sequence of DNA without unwinding it
(e.g. a restriction enzyme).
restriction enzyme EcoR V
DNA replication occurs with great fidelity
Somatic cell DNA stability and reproductive-cell DNA
stability are essential. Why?
Identity
Genetic diseases
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
• In eukaryotes , replication only occurs during the S
phase of the cell cycle.
• Replication rate in eukaryotes is slower resulting in a
higher fidelity/accuracy of replication in eukaryotes
The mechanism of DNA replication
Arthur Kornberg et al (Nobel 1959)
He was the first to discover how molecules of deoxyribonucleic acid (DNA) duplicate within bacterial cells and also the first to devise a method for synthesizing this process in a laboratory setting.
For these achievements he shared the 1959 Nobel Prize in physiology or medicine with Severo Ochoa.
REPLICATION STEPS• Initiation
• Proteins bind to DNA and open up double helix
• Prepare DNA for complementary base pairing
• Elongation
• Proteins connect the correct sequences of nucleotides into a continuous new strand of DNA
• Termination
• Proteins release the replication complex
Basic rules of replication
A. Semi-conservative
B. Starts at the ‘origin’
C. Can be uni or bidirectional
D. Semi-discontinuous
E. Synthesis always in the 5-3’ direction
F. RNA primers required
DNA replication Of the 3 possible models, replication is…
A) Semi-
conservative
Meselson and Stahl experiment
• Meselson and Stahl concluded that DNA employs semi-conservative replication. They placed the bacteria in an environment which contained a selected Nitrogen isotope.
• They first used N14. The bacteria then integrated this isotope into their DNA. Later they used an environment that contained N15.
• They then looked at which of the isotopes the bacterial DNA contained.
• They found that it contained both isotopes of nitrogen, which implies that conservative replication is not the correct conclusion
Semiconservative replication
Half the original DNA molecule is
saved, or conserved in the daughter
molecules. This is why the process
is called semi-conservative.
B) Starts at originInitiator proteins identify specific base sequences on DNA called sites of origin
Prokaryotes – single origin site E.g E.coli - oriC
Eukaryotes – multiple sites of origin (replicator)
E.g. yeast - ARS (autonomously replicating sequences)
Prokaryotes Eukaryotes
C) Uni or bidirectionalReplication forks move in one or opposite directions
D) Semi-discontinuous replication
Anti parallel strands replicated simultaneously
Leading strand synthesis continuously in 5’– 3’
Lagging strand synthesis in fragments in 5’-3’
Semi-discontinuous replication
New strand synthesis always in the 5’-3’ direction
E) Synthesis is ALWAYS in the 5’-3’ direction
Nucleotide recognition
Enzyme catalysed polymerisation (DNA polymerase)
Complementary base pair copied
Substrate used is dNTP
Where does energy for addition
of nucleotide come from?
What happens if a base
mismatch occurs?
DNA polymerase has 3’ 5’ exonuclease activity in order to correct errors
From cleavage of high energy phosphate of incoming triphosphate
PROOFREADING
• The term proofreading is used in genetics to refer to the error-correcting processes,.
• When an incorrect base pair is recognized, DNA polymerase reverses its direction by one base pair of DNA and excises the mismatched base. Following base excision, the polymerase can re-insert the correct base and replication can continue.
F) RNA primers required• DNA polymerase can only join an incoming nucleotide to one that is base-paired
• RNA primase provides a base paired 3’ end as a starting point for DNA pol by synthesising ~10 nucleotide primers
Core proteins at the replication fork
- regulate the overwinding or underwinding of DNA
- separates 2 strands
- RNA primer synthesis
- synthesis of new strand
- seals nick via phosphodiester linkage
Topoisomerase
Helicases
Primase
DNA polymerase
DNA ligase