DNA Replication
Synonyms: Doubling, Duplication,
Copying, Biosynthesis and
Synthesis
From Monomers to Polymers
Complementary
surfaces
Watson-Crick
base pairs
DNA Replication
• Replication of DNA occurs during the
process of normal cell division cycles.
• The DNA of the daughter cells must be the
same as the parental cell.
• DNA replication must possess a very high
degree of fidelity.
• The entire process of DNA replication is
complex and involves multiple enzymatic
activities.
DNA Synthesis
• Biochemical process of making new DNA
strand from nucleotides using various
enzymes and a DNA molecule as a template.
• All DNA replication uses DNA synthesis, but
not all DNA synthesis need give complete
DNA replication of DNA.
• Sometimes referred to a “biosynthesis” since
you can make “synthetic” DNA using a
synthesis machine and chemicals.
Template Replication Model
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The two strands could separate from one
another, each still containing the complete
information, and synthesize a new strand.
Did the two strands unwind and each act as
a template for new strands?
Did the strands not unwind, but somehow
generate a new double stranded DNA copy
of entirely new molecules?
Or was there some combination of both?
A Classic Experiment
• In 1957 Messelson and Stahl added
heavy nitrogen (15N) to bacteria and
arrested growth at various times.
• Heavier 15N DNA could be separated
from lighter 14N DNA in Cesium Chloride
gradient centrifugation.
• This was a very important method used
in other experiments.
(a) is a photograph of the tube under UV
light.
(b) is a graph of darkness of each band
Results of the Experiment
• Results show that after one generation,
the double stranded DNA is 1/2 heavy
(from the parent) and 1/2 light (newly
synthesized).
• This result is predicted by
semiconservative replication.
• Conclusion- (as predicted by Watson
and Crick) DNA strands serve as
templates for their own replication.
Biochemical Mechanism of
DNA Replication
• DNA replication is not a passive and
spontaneous process.
• Many enzymes are required to unwind
the double helix and to synthesize a
new strand of DNA.
Biochemical Mechanism of
DNA Replication
• Enzymes are biochemical catalystsThey speed up a reaction without being
consumed by the reaction.
• Enzymes break and form covalent
bonds.
• Every chemical event in the cell
depends on the action of an enzyme.
Studying DNA Replication
• In Vitro Extracts: Biochemists break open
cells and “extract” their contents.
• They use biochemical techniques to separate
the proteins based on size and charge as well
as their ability to bind to DNA.
• “ A biochemist devoted to enzymes could, if
persistent, reconstitute any metabolic event in
the test tube as well as the cell does it.”
Arthur Kornberg Nobel Prize for DNA
Replication.
DNA Polymerase I Discovery
• Kornberg decided to study bacteria, reasoning
that they replicated DNA rapidly. Whatever
enzymes was required must be present in
simple bacterial cells.
• He took rapidly growing bacteria and broke
them open.
• The DNA replication enzymes were separated
into fractions and each fraction was assayed
to identify the DNA polymerase enzyme.
Studying DNA Replication
• Scientists need an assay or test to determine
if their extracts are making DNA.
• Basic assay for DNA replication is to add
various DNAs to a tube with extracts,
nonradioactive dNTPs and one radioactive
dTTP.
• Separate the small dNTPs from the bigger
radioactive DNA pieces and measure
radioactivity incorporated.
Pulse-Chase Assays
• Add the radioactively labeled molecules
for a short time period (pulse).
• Add a large amount of nonradioactive
(cold) molecules of the same sort after
the short time (chase).
• Molecules synthesized during the pulse
will be labeled and chase molecules will
not be labeled.
Combining In Vitro Extracts
With Genetics
• Kornberg combined biochemistry and
genetics to study DNA replication.
• Isolated "conditional mutant bacteria"
which were temperature sensitive;
• Temperature sensitive bacteria made
DNA and replicated the bacterial
chromosome at 37° C (permissive) but
couldn’t at 42° C (restrictive).
Two Kinds of Conditional
Mutants
• Quick stop mutants at a shift to restrictive
temperature, DNA synthesis stopped
immediately along with replication of the
bacterial chromosome. This was due to a
malfunction in the elongation machinery itself
or in the enzymes required for essential
precursors
• Slow stop mutants at a shift to restrictive
temperature, the cell is able to finish
replication of the bacterial chromosome but
couldn’t begin another round.
In Vitro Complementation
• If normal enzyme extract is added to a
template DNA, replication occurs.
• If an extract from conditional mutants cells is
added to a template, no replication occurs.
• If this same mutant extract is added along with
purified normal proteins, replication can take
place if the new purified enzyme replaces the
mutant enzyme.
Kornberg’s Success
• Kornberg was awarded the Nobel Prize
in Medicine in 1959.
• http://www.nobel.se/medicine/laureates/
1959/
Mutation Analysis Reveals
Polymerase I Can’t Be the
Only DNA Polymerase
• These types of heat sensitive mutants
revealed that Kornberg’s Polymerase I could
be mutated. The bacterial cells were weak
growers but still lived.
• Scientists reasoned there must be more to
the situation.
• No, he didn’t have to give back the Nobel
Prize….
The real situation is more
complex
• Polymerase III is the critical DNA
polymerase.
• Polymerase I is an important repair
enzyme
New DNA strands are always synthesized in the 5' to
3' direction. The 5' triphosphate can only be added to
a free 3'OH of deoxyribose.
Why Does DNA Polymerase
Only Add Nucleotides to 3‘ OH?
• A triphosphate is required to provide
energy for the bond between a newly
attached nucleotide and the growing
DNA strand.
• However, this triphosphate is very
unstable and can easily break into a
monophosphate and an inorganic
pyrophosphate, which floats away into
cell.
Why Does DNA Polymerase
Only Add Nucleotides to 3'?
• At the 5' end of the DNA, this triphosphate can
easily break. If a strand has been sitting in the
cell for a while, it would not be able to attach
new nucleotides to the 5' end once the
phosphate had broken off.
• The 3' end only has a hydroxyl group, so as
long as new nucleotide triphosphate are always
brought by DNA polymerase, synthesis of a
new strand can continue no matter how long
the 3' end has remained free.
This Presents a Problem
• One strand of the double helix is 5' to 3'
and the other one is 3' to 5'.
• How can DNA polymerase synthesize
new copies of the 5' to 3' strand, if it can
only travel in one direction?
DNA Synthesis Occurs at
Replication Forks.
• Synthesis is bidirectional from a
replication origin (or origins).
• Replication forks are highly organized
structures in cells (DNA synthesis
"machines").
• What is going on in the fork? Several
models were proposed.
•The two antiparallel strands are replicated simultaneously
•in both directions and semi-discontinuously.
•RNA primers are used to initiate a new strand.
•DNA polymerase uses the RNA primers as starting points to
•synthesize progeny DNA strands.
• Both strands are replicated 5’ to 3’ direction.
Pulse Chase
Experiments reveal DNA
is made in short pieces
that become longer
when combined.
Adding Other Enzymes to In
Vitro Extracts
• RNases degrade RNA.
• DNases degrade DNA. They can be
single or double-stranded DNase.
• Make an In Vitro extract and add these
enzymes. This showed Kornberg and
others that there could be no replication
if there was not RNA made.
Other Proof RNA Is Involved
• Radioactive UTP is added to the labeled
fragments and over time in a pulse
chase assay, removed.
Helicase Function
• Helicase unwinds duplex DNA at the
replication fork. DnaB helicase is a 5' to 3'
helicase that travels along the lagging single
strand as it unwinds upstream duplex DNA.
• This creates two antiparallel DNA single
strands.
• The leading DNA strand is the template for
DNA polymerase III holoenzyme, which
synthesizes a continuous strand.
Primase Makes the RNA
Primers
• The function of primase is to initiate, or prime,
DNA synthesis.
• Primase is required because DNA polymerases
cannot initiate polymer synthesis; DNA
polymerases can only elongate polymers that
have already been initiated.
• RNA polymerases can initiate polymer
synthesis in addition to being able to elongate.
There are two RNA polymerases in E. coli and
the one involved in DNA replication is called
primase.
Electron Micrographs and
Pulse Chase
• Label the molecules with short burst of
radioactivity.
• Take electron micrograph pictures of
DNA molecules and look at where the
radioactivity is.
• Use short circles of DNA called
plasmids.
How Does the Enzyme
Complex Know Where to Start
Replication?
Origin of Replication
• Strand separation occurs at what is
termed an "origin of replication".
• Bacteria typically have a single origin of
replication. Eukaryotes have many
origins of replication in each
chromosome.
• Viruses have an origin of replication.
• Abbreviation is ORI.
OBR= Origin or Bidirectional
Replication = ORI
• Genetically defined origins of replication (ori)
are DNA sequences that are necessary and
sufficient for initiation of replication
• In most cases, the molecularly defined OBR's
and the genetically defined oris coincide. A
few cases exist, however, where an ori
causes bidirectional replication to initiate in
an adjacent region.
Bidirectional Circular DNA Replication
ORIs
• DnaB can’t find the origin of replication by
itself; it needs some help
• DnaA binds to the specific sequences that
define the origin (oriC) and separates the
strands yielding an open complex
• DnaC delivers DnaB to this template
• One DnaB hexamer clamps around each
single strand forming the pre-priming complex
Terminators
• Terminators function unidirectionally and stop
replication as the fork passes through those
sequences.
• The second sequence of the pair is thought to
act as a back-up, in case the first fails.
• When two forks collide, either between the
two pairs of termination signals, or at a
termination signal, the forks are annihilated.
Concatenated circles should result.
Electron Micrograph
Information
• The replication bubble enlarges as
replication proceeds.
• The double-stranded DNA template is
not completely denatured before DNA
synthesis begins.
• Unwinding and synthesis are coupled.
• This is indicated by the uniformly equal
thicknesses of the bubble walls.
Electron Micrograph
Information
• The DNA template at the replication fork must
be unwound to allow access by the DNA
polymerase and DNA synthesis.
• An enzyme activity is needed to relieve
superhelical stress that results from the
unwinding process.
• Otherwise, the newly replicated bubble would
be extensively interwound.
DNA Replication: Important
Points
• DNA polymerases cannot melt or unwind
duplex DNA- they need help from helicases
and gyrases in vivo or heating in vitro.
• DNA polymerases cannot initiate chains,but
can only extend a pre-existing DNA or RNA
strand.
• DNA replication can only start at an origin of
replication.
• All DNA strands grow in a 5’ to 3’ direction
during replication.
The central dogma of molecular biology:
DNA makes RNA makes Protein is also violated by
some viruses that use RNA to make DNA.
• In 1975 Howard Temin was given Nobel
Prize for discovery of the enzyme
reverse transcriptase which used RNA
as a template to make DNA in certain
viruses.
• http://www.nobel.se/medicine/laureates/
1975/
Retroviruses have a single stranded RNA
genome that is converted to double
stranded DNA in three steps:
• RNA is the template for RT-catalyzed
synthesis of an RNA-DNA duplex.
• The RNA is degraded by the RNaseH
activity of the viral RT.
• The DNA is copied (by host cell
enzymes) to produce double stranded
DNA. In this form, the retroviral genome
can be inserted into the cellular
chromosomes.
Host-cell RNA polymerase is used to
make virus-related RNA.
These RNA strands serve as
templates for making new copies of
the viral chromosomal RNA and serve
also as mRNA.
Reverse Transcriptase lacks a
proofreading 3' --> 5' exonuclease.
“Error-prone replication" leads to
mutations in all HIV-1 genes.
The rapidly changing genome that
results, is a difficult moving target to
hit with therapeutic drugs