Replication
Replication
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Basic Molecular Biology: The Genetic Process
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Basic Molecular Biology: The Genetic
Process
Replication
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Learning objectives
Understand the basic rules governing DNA replication
Understand the function of key proteins involved in a
generalised replication model
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`It has not escaped our notice that the specific
pairing we have postulated immediately
suggests a possible copying mechanism for
the genetic material’
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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
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DNA stores information in the sequence of its bases
restriction
enzyme EcoR V 9
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
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THE EUKARYOTIC CELL CYCLE
S phase
DNA synthesis
G1
Growth phase 1 Main checkpoints
G2
Go M
Growth phase 2
Quiescent Mitosis
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Three main features of the DNA synthesis reaction:
• Energy for this reaction is derived from the release of two of the three
phosphates of the dNTP.
2. DNA polymerase “finds” the correct complementary dNTP at each step in the
lengthening process.
3. Direction of synthesis is 5’ to 3’
DNA polymerase
Image credit:
Protein Data Bank
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The mechanism of DNA replication
– 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
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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
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DNA replication
Of the 3
possible
models,
replication is…
A) Semi-
conservative
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B) Starts at origin
Initiator proteins identify specific base sequences on DNA
called sites of origin
Prokaryotes Eukaryotes
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C) Uni or bidirectional
Replication forks move in one or opposite directions
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D) Semi-discontinuous replication
Anti parallel strands replicated simultaneously
Leading strand synthesis continuously in 5’– 3’
Lagging strand synthesis in fragments in 5’-3’
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D) Semi-discontinuous replication
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Origin of replication (e.g., the prokaryote example):
~245 bp in E. coli
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Initiation of replication, major elements:
Initiator proteins and DNA helicase binds to the DNA at the replication fork and
untwist the DNA using energy derived from ATP (adenosine triphosphate).
(Hydrolysis of ATP causes a shape change in DNA helicase)
The RNA primer is removed and replaced with DNA by polymerase I, and the
gap is sealed with DNA ligase.
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DNA replication is continuous on the leading strand and semidiscontinuous on the
lagging strand:
The two DNA strands are of opposite polarity, and DNA polymerases only synthesize
DNA 5’ to 3’.
continuous
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(SSB)
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Replication Enzymes
OH 3’
5’
3’ 5’
OH 3’
5’
3’ 5’
OH 3’
5’
3’ 5’
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DnaB
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In prokaryotes, there are three main types of DNA polymerase
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Eukaryotic enzymes:
Five common DNA polymerases from mammals.
5’ SSB Proteins
Okazaki Fragments
1 ATP
Polymerase III 2
Helicase
Lagging strand 3 +
Initiator Proteins
3’
primase base pairs
Polymerase III 5’
3’
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DNA ligase seals the gaps between Okazaki fragments with a
phosphodiester bond
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Model of DNA Replication
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Model of DNA replication
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Model of DNA replication
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DNA replication in eukaryotes
Copying each eukaryotic chromosome during the S phase of the cell cycle presents
some challenges:
2. Cell will not enter the mitotic phase unless all the DNA has replicated.
4. Checkpoints in the system include proteins call cyclins and enzymes called cyclin-
dependent kinases (Cdks).
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• Each eukaryotic chromosome is one linear DNA double helix
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What about the ends (or telomeres) of linear chromosomes?
DNA polymerase/ligase cannot fill gap at end of chromosome after RNA primer is
removed. If this gap is not filled, chromosomes would become shorter each
round of replication!
Solution:
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Core proteins at the replication fork
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Core proteins at the replication fork