dna stands for deoxyribose nucleic acid · • dna replication 5’->3’ • dna proof reading...
TRANSCRIPT
DNA stands for deoxyribose nucleic acid
This chemical substance is present in the nucleus
of all cells in all living organisms
DNA controls all the chemical changes which
take place in cells
DNA
DNA Structure
• Watson & Crick 1953
– Proposed the double helix shape of DNA and
received a Nobel Prize
• Maurice Wilkins and Rosalind Franklin
– Produced images of DNA using x-ray
diffraction
– Franklin passed before Nobel Prize was
awarded
Four requirements for DNA to be genetic material
Must carry information
– Cracking the genetic code
Must have the ability to be reproduced
– DNA replication
Must allow for information to change
– Mutation
Must govern the expression of the phenotype
– Gene function
DNA is a very large molecule made up of a long
chain of sub-units
The sub-units are called nucleotides
Each nucleotide is made up of
a sugar called deoxyribose
a phosphate group -PO4 and
an organic base
DNA molecule
Ribose is a sugar, like glucose, but with only five
carbon atoms in its molecule
Deoxyribose is almost the same but lacks one
oxygen atom
Both molecules may be represented by the symbol
Ribose & deoxyribose
The most common organic bases are
Adenine (A)
Thymine (T)
Cytosine (C)
Guanine (G)
The bases
The deoxyribose, the phosphate and one of the bases
adenine
deoxyribose
PO4
Combine to form a nucleotide
Nucleotides
A molecule of
DNA is formed
by millions of
nucleotides
joined together
in a long chain
PO4
PO4
PO4
PO4
sugar-phosphate
backbone+ bases
Joined nucleotides
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
2-stranded DNA
The bases always pair up in the same way
Adenine forms a bond with Thymine
and Cytosine bonds with Guanine
Bonding 1
Adenine Thymine
Cytosine Guanine
PO4
PO4
PO4
thymine
PO4
PO4
PO4
PO4
adenine
cytosine
PO4
guanine
Bonding 2
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
Pairing up
A
The paired strands are coiled into a spiral called
A DOUBLE HELIX
sugar-phosphate
chain
bases
THE DOUBLE
HELIX
Before a cell divides, the DNA strands unwind
and separate
Each strand makes a new partner by adding
the appropriate nucleotides
The result is that there are now two double-
stranded DNA molecules in the nucleus
So that when the cell divides, each nucleus
contains identical DNA
This process is called replication
Replication overview
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
The strands
separate
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
Each strand builds up its partner by adding
the appropriate nucleotides
Overview of Processes
• DNA replication 5’->3’
• DNA proof reading
• Lagging strand, back-stitching, Okazaki fragment
• Proteins involved:1. DNA polymerase,
primase
2. DNA helicase and single-strand DNA-binding protein (SSB)
3. DNA ligase, and enzyme to degrade RNA
4. DNA topoisomerases
5’ and 3’
ATP
dATP
Semi-Conservative DNA Replication
Step 1:
• Untwisting and Unzipping
• Helicase separates the strands by un-pairing
the bases
• How can you remember helicase?
• Replication Fork- Y-shaped region where
the parental strands of DNA are being
unwound
• Leading Strand- will be replicated
continuously
• Lagging Strand- will be replicated in
sections
• Template Strands- ?
Step 2
• Stabilization
• Single-Strand Binding Proteins- will bind to
unpaired DNA strands to keep them stable
Step 3
• Maintenance
• Topoisomerase- enzyme that breaks,
swivels and rejoins parental DNA strands
Step 4
• Initiation
• The enzymes that synthesize DNA cannot
start without a guide
• Primase- an enzyme that adds a strip of
RNA nucleotides to the parental strands
• This strip is approximately 5-10 nucleotides
long and is referred to as the Primer
• RNA Primers- point of initiation of DNA
replication
– Base pairs or nucleotides are similar with the
exception of Uracil.
– Uracil replaces Thymine and so, pairs with
Adenine
Uracil
Step 5
• DNA Synthesis
• DNA Polymerase III- starts at the primer,
adding bases in only one direction
• DNA Polymerase moves from the 5’ end
toward the 3’ end
Caveats of Elongation
• DNA has a defined 5’ and 3’ end which
gives it directionality
• DNA exists as antiparallel strands which
means the new strands must also be
antiparallel
• What’s the issue?
Leading Strand
• This template strand is called as such
because it is replicated continuously
• The DNA Polymerase binds to replication
fork at the primer and simply adds free
nucleotides to the leading strand until it has
completely synthesized a complimentary
strand.
Lagging Strand
• For the second template strand it is a bit
more complicated
• The DNA Polymerase must synthesize the
complimentary strand working away from
the replication fork
• **this must also be synthesized 5’ to 3’
• It is synthesized discontinuously
• Okazaki Fragments- the segment of the
lagging strand (100-200bp in eukaryotes)
Lagging Strand
• Thus, several primers are needed on the
lagging strand
• DNA Polymerase I- enzyme that will
replace the RNA primers
– Adds to the 3’ end of each Okazaki Fragment
– Exonuclease- detaches the RNA primers
• DNA Ligase- joins the sugar-phosphate
backbones of the strands together to make a
continuous strand
https://www.youtube.com/watch?
v=TNKWgcFPHqw
DNA Repair
• Errors occur during replication
• There is approximately 1 error in every 10
billion nucleotides after replication
• However, errors due occur during the
process and more frequently
– 1 in 100,000 bps
Proofreading
• While the base pairs are being added, DNA
Polymerase check to make sure they are
added correctly
• It is not a flawless proofread and correction
of these nucleotides is referred to as
mismatch repair
Proofreading
• Nucleotides can be incorrectly paired or
even altered after replication
• DNA has to frequently be repaired
• Chemical or physical agents can lead to
alterations of bps and warrant repair
• Reactive chemicals, radioactive emissions,
X-rays, UV rays and other molecules can
lead to nucleotides changes
– Often, these changes are repaired before
mutations can occur
Nuclease
• An enzyme called a nuclease will excise the
altered nucleotides
• Then other enzymes will replace those
nucleotides with the correct bps, and then
make the strand whole again
• This is done by using the unaffected strand
of DNA as a template
• This is an example of nucleotide excision
repair
Application
• UV light can lead to skin cancer
• UV light can catalyze the production of
thymine dimers
• This causes a covalent bond to form
between thymine nucleotides
Xeroderma Pigmentosum
• Xeroderma pigmentosum (XP) is a rare
autosomal recessive genetic disorder of
DNA repair in which the ability to repair
damage caused by ultraviolet (UV) light is
deficient
Problems with Replication
• Over the course of several replications, the
DNA faces some changes
• The Strands of DNA actually become
shorter
Telomeres
Prokaryote VS Eukaryote DNA Replication
Eukaryotes
• X-shaped Chromosome
• Semi-Conservative
• Replicates in Nucleus
• Many points of Origin
• Two templates
• Leading and Lagging
Strands
• At least 11 major
Polymerases
• 50 nucleotides/sec
Prokaryotes
• Ring-shaped Chromosome
• Semi-Conservative
• Replicates in Cytoplasm
• One point of origin
• One template
• Bidirectional and
Continuous
• 2 major polymerases
• 500 nucleotides/sec
Prokaryote DNA Replication
Prokaryote VS Eukaryote DNA Replication