dna
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GUMEDE ESTHER NTOMBIFUTHI 3RD YEAR
UNIVERSITY OF JOHANNESBURG 2014
NUCLEIC ACIDS :
DNA StructureDNA consists of two molecules that are
arranged into a ladder-like structure called a Double Helix.
A molecule of DNA is made up of millions of tiny subunits called Nucleotides.
Each nucleotide consists of:1. Phosphate group2. Pentose sugar-Deoxyribose3. Nitrogenous base
Nucleotides
Phosphate
Pentose
Sugar
Nitrogenous
Base
DNA Structure Helps Explain How It Duplicates
DNA is made up of two nucleotide
strands held together by hydrogen
bonds
Hydrogen bonds between two strands
are easily broken
Each single strand then serves as
template for new strand
DEOXYRIBONUCLEIC ACID
\DNA usually exists as a double-stranded structure, with both strands coiled together to form the characteristic double-helix.
Each single strand of DNA is a chain of four types of nucleotides having the bases:
AdenineCytosineGuanineThymine
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NucleotidesThe phosphate and sugar form the
backbone of the DNA molecule, whereas the bases form the “rungs”.
There are four types of nitrogenous bases.
Orientation of DNA
The directionality of a DNA strand is due to the orientation of the phosphate-sugar backbone.
The carbon atoms on the sugar ring are numbered for reference. The 5’ and 3’ hydroxyl groups (highlighted on the left) are used to attach phosphate groups.
Nucleotides
A
Adenine
T
Thymine
G
Guanine
C
Cytosine
NucleotidesEach base will only bond with one
other specific base.
Adenine (A)Thymine (T)
Cytosine (C)Guanine (G)
Form a base pair.
Form a base pair.
DNA StructureA gene is a section of DNA that codes
for a protein.
Each unique gene has a unique sequence of bases.
This unique sequence of bases will code for the production of a unique protein.
It is these proteins and combination of proteins that give us a unique phenotype.
Protein
DNA
Gene
Trait
A Nucleoside is a combination of Pentose sugar & Nitrogen
Base
A Nucleotide is a combination of
nucleoside & Phosphoric Acid
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P
A
P C
P
G
P T
P C
P
G
P
A
PC
PT
G
P
PC
P
A sugar and phosphate “backbone” connects nucleotides in a chain.
P
G
P
Two nucleotide chains together wind into a helix.
DNA strands are antiparallel.
DNA has directionality.
5’
3’
3’
5’
Hydrogen bonds between paired bases hold the two DNA strands together.
DNA BACKBONE
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Nucleotides are matched between strands through hydrogen bonds to form base pairs. Adenine pairs with thymine
and cytosine pairs with guanine
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These terms refer to the carbon atom in deoxyribose
to which the next phosphate in the chain
attaches. Directionality has consequences in DNA
synthesis, because DNA polymerase can synthesize DNA in only one direction by adding nucleotides to
the 3' end of a DNA strand.
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The pairing of bases in DNA through hydrogen bonding means that the information contained within each
strand is redundant. The nucleotides on a single strand can be used to reconstruct nucleotides
on a newly synthesized partner strand.
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FunctionsDNA is used to store genetic
informationIt is replicated before cell division
DNA is very important so it is stored in the nucleus.
It never leaves the nucleusYour DNA stores the code for your
proteins, which exhibit your “traits” The DNA gets converted to RNA in
order to move out into the cytoplasm
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DNA replication is a biological process that occurs in all living organisms and copies their exact DNA. It is the basis
for biological inheritance.
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Each old Each old
strand stays strand stays
intact intact Each new DNA Each new DNA
molecule is molecule is
half “old” and half “old” and
half “new”half “new”Fig. 1-7, p.212
The first major step for the DNA Replication to take place is the
breaking of hydrogen bonds between bases of the two
antiparallel strands.
The unwounding of the two strands is the starting point. The splitting happens in places of the chains
which are rich in A-T. That is because there are only two bonds between Adenine and Thymine. There are three hydrogen bonds between Cytosine and Guanine.
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Helicase is the enzyme that splits the two
strands. The structure that is created is known as "Replication Fork".
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In order for DNA replication to begin, the double stranded DNA helix must first be opened. The sites
where this process first occurs are called replication origins. Helicase unwinds the two single strands
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Binding proteins prevent single strands from rewinding.
Replication
Helicase protein binds to DNA sequences called origins and unwinds DNA strands.
5’ 3’
5’
3’
Primase protein makes a short segment of RNA complementary to the DNA, a primer.
3’ 5’
5’ 3’
Overall directionof replication
5’ 3’
5’
3’
5’
3’
3’ 5’
DNA polymerase enzyme adds DNA nucleotides to the RNA primer.
5’
5’ 3’
5’
3’
3’
5’
3’Overall directionof replication
Leading strand synthesis continues in a 5’ to 3’ direction.
3’ 5’ 5’
5’ 3’
5’
3’
3’
5’
3’Overall directionof replication
Okazaki fragment
Leading strand synthesis continues in a 5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
Replication Fork
The replication fork is a structure that forms within the nucleus during
DNA replication. It is created by helicases, which break the hydrogen bonds holding the two DNA strands
together. The resulting structure has two branching "prongs", each one
made up of a single strand of DNA. These two strands serve as the
template for the leading and lagging strands, which will be created as
DNA polymerase matches complementary nucleotides to the templates; The templates may be properly referred to as the leading strand template and the lagging
strand template
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DNA polymerase enzyme adds DNA nucleotides to the RNA primer.
5’
5’
5’
3’
5’
3’
3’
3’
DNA polymerase proofreads bases added and replaces incorrect nucleotides.
3’
5’
3’
5’
5’ 3’
5’
3’
3’
5’ 5’ 3’
Leading strand synthesis continues in a 5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
5’ 5’
5’ 3’
5’
3’
3’
5’
3’
3’
Leading strand synthesis continues in a 5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
Okazaki fragment
5’
5’ 3’
5’
3’
3’
5’
3’
3’
5’ 5’ 3’
Leading strand synthesis continues in a 5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
5’
5’
3’
3’
5’
3’
5’ 3’
5’
3’
3’
5’
Exonuclease enzymes remove RNA primers.
Exonuclease enzymes remove RNA primers.
Ligase forms bonds between sugar-phosphate backbone.
3’
5’
3’
5’ 3’
5’
3’
3’
5’
One of the most important steps of DNA Replication is the binding of RNA Primase in the initiation point of the 3'-5' parent chain.
RNA Primase can attract RNA nucleotides which bind to the DNA nucleotides of the 3'-5' strand due to the hydrogen bonds between the bases. RNA nucleotides are the primers (starters) for the binding of DNA nucleotides.
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In the lagging strand the DNA Pol I-Exonuclease- reads the fragments and removes the RNA Primers. The gaps are closed with the action of DNA Polymerase which adds complementary nucleotides to the gaps and DNA Ligase which acts as a glue to attach the phosphate to the sugar by forming phosphodiester bond.
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Enzymes in ReplicationEnzymes (Helicases) unwind the two
strands
DNA polymerase needed for the synthesis of complementary strand
DNA ligase joins pieces of the lagging strand together
Helicase unwinds parental double helix
Binding proteinsstabilize separatestrands
DNA polymerase binds nucleotides to form new strands
Ligase joins Okazaki fragments and seals other nicks in sugar-phosphate backbone
Primase adds short primer to template strand
Exonuclease removesRNA primer and inserts the correct bases
DNA Replication models
There are three possible models that describe the accurate creation of the daughter chains:
Semiconservative Replication Conservative Replication Dispersive Replication
Figure 11.211-6
Replicatoin can’t just start:
1. 1. All DNA polymerases need a primerAll DNA polymerases need a primer
22. The primer can be a piece of RNA or DNA. The primer can be a piece of RNA or DNA
3. 3. It must be “base-paired” with the It must be “base-paired” with the “template” and with “template” and with 3’OH3’OHThus:Thus:
3’ 5’5’ 3’
Template strandTemplate strand
primerprimer
Synthesis directionSynthesis direction
““Proof-reading” is essential at DNA replicationProof-reading” is essential at DNA replication
•What is proof-reading?What is proof-reading?
11. If there is a wrong base built in, . If there is a wrong base built in, then there is no base paring possiblethen there is no base paring possible..
2. The DNA polymerase can’t 2. The DNA polymerase can’t continue on building in the next continue on building in the next base.base.
3. The DNA polymerase removes the “wrong” base3. The DNA polymerase removes the “wrong” base and starts overand starts over
Klenow Fragment (of pol I)(The proof-reading activity)
Bacterial DNA polymerases may vary in their Bacterial DNA polymerases may vary in their subunit compositionsubunit composition However, they have the same type of catalytic However, they have the same type of catalytic
subunitsubunitStructure resembles a
human right hand
Template DNA thread through the palm;
Thumb and fingers wrapped around the DNA
Proteins involved in E. coli replication
Terms to be known
DNA replication mistakesThe most errors in DNA sequence occur
during replication.
Reparation takes place after replication is finished
DNA polymerases can get the right sequence from the complementary strand and repair, along with DNA ligase, the wrong bases.
Is a method in which multiple repetitions of DNA replication are performed in a test tube.
Mix in test tube:
DNA template DNA to be amplified
Primers one complementary to each strand
Nucleotides dATP,d GTP, dCTP, and dTTP
DNA polymerase heat stable form from thermophilic bacteria
DNA template is denatured with heat to separate strands.
C T T G A T CGC
3’5’
G ATCAA GCG
3’ 5’
DNA template is denatured with heat to separate strands.
C T T G A T CGC
3’5’
G ATCAA GCG
3’ 5’
C T T G A T CGC
3’5’
G ATCAA GCG
3’ 5’
Each DNA primer anneals, binding to its complementary sequence on the template DNA
C T T
GCG
5’5’
3’3’
DNA polymerase creates a new strand of DNA complementary to the template DNA starting from the primer.
C T T G A T CGC
3’5’
G ATCAA GCG
3’ 5’
C T T
GCG
5’5’3’
C G CG A T
G A A C T A
3’
Denaturation
Each DNA primer anneals, bindingto its complementary sequenceon the template DNA
DNA template is denatured with high heat to separate strands.
Annealing
Extension DNA polymerase creates a new strand of DNA complementaryto the template DNA starting from the primer.
Multiple rounds of denaturation-annealing-extension areperformed to create many copies of the template DNA between the two primer sequences.
Visualization of PCR products
agarose gel-electrophoresis of DNATop
Bottom
Slab of agarose gel (ethidium bromide staining)
Positive pole
Negative pole
DNA is Negatively charged
Small molecules dissolve faster
separation based on size
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