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11/9/16
The 4 S’s
Size
Solubility
Shape
Stability
Tautomeric Forms of Bases
Uracil
(keto)
Cytidine
(amine)
⥄ Uracil
(enol)
⥄ Cytidine
(imine)
D
A
H
N
If during replication, an A
is in the imino tautomer,
if will be complementary
to the pyrimidine C
rather than T…...
N
N H
N
N
R
D
H N
A
O
N
N
H
O
H
D
A
R
H
N
N
N
R
A
N
H
H
A
D
N
O
N
O
N
H
H N
D
A
T
O
N
N H
N
R
R
D
G
N
N
A = ACCEPTOR H
D = DONOR
A
N
C
N
R
O
H
A
D
H
N
N
N
R
N
H
O
H
N
N
N
O
R
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Tautomeric Forms of Bases
Mutagenesis
Imino tautomer of A
*
5’-GGGTTTATTTGGG-3’
3’-CCCAAATAAACCC-5’
Replication
GGGTTTATTTGGG
CCCAAATAAACCC
GGGTTTATTTGGG
CCCAAACAAACCC
Replication
GGGTTTGTTTGGG
CCCAAACAAACCC
GGGTTTATTTGGG
CCCAAATAAACCC
Tautomeric Forms of Bases
Modified bases can stabilize the rare tautomer
enol tautomer of U
D
A
H
H N
(enol)
O
N
N
N
H
H
D
D
A
N
N H
N
R
D
A
N
R
O
H
N
N
N
R
A
N
A
H
H
A
D
N
O
N
O
N
H
H N
D
A
T
O
N H
N
R
N
D
G
N
N
R
H
A
N
C
N
R
O
H
A
D
H
N
N
N
R
N
H
O
H
N
N
N
O
R
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Acid/base Properties of Bases
At pH 7, all bases are neutral. They can fit into middle of B-DNA without
repulsions
But, acidic or basic treatment will protonate or deprotonate these bases.
At low pH, N7 of guanine, N1
of adenine, and N3 of cytosine
become protonated.
At high pH, N1 of Guanine and
N3 of Uracil (Thymine)
become deprotonated.
Acid/base Properties of Bases
Charged bases can de-stabilize the glycosidic bond
and mutagenesis
pKa 2.4
Destabilizes
glycosidic bond,
leads to “AP-site” *
N
O
N
R
N
N
N
H
H
N
H
pKa 3.8
H
D
H
N
H
N
N
N
R
H
D
Leads to A à G
mutations
A
O
O
N
N
R
O
H
* “AP-site” refers to a-purinic or a-pyridinic site
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Other Chemical Changes of Bases
DNA Damage:
• oxidation
• alkylation
• degradation
Other Chemical Changes of Bases
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Other Chemical Changes of Bases
Deamination of C
yields U, which leads to
C à T mutations
Other Chemical Changes of Bases
Point-mutation by Oxidation
Deamination of C
yields U, which leads to
C à T mutations
Deamination of A yields
Hyp, which leads to A
à G mutations
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Other Chemical Changes of Bases
8-Oxoguanine favors
the syn conformation,
which leads to O8-G:A
bp (G à T mutations)
Other Chemical Changes of Bases
and other bases
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Other Chemical Changes of Bases
DNA Methylation
Some methylations are damaging.
•
Methylation of G at O6 can basepair with C or T, which causes G
à A mutations
Some methylations are important.
• Methylation for endonuclease restriction recognition or protect DNA from digestion by
endonuclease.
• Methyl group lie in the major groove and can be used in the interaction with DNA interaction
proteins.
• Importance of DNA methylation in replication: it is used to differentiate between the new and
old strand. If there is a mutation, the repairing system will use the methylated strand as the
template.
• In mammalian system, the promoter region has regular CpG content. Methylation at these
sites can switch off eukaryotic gene expression.
Other Chemical Changes of Bases
Alkylating Agents
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Other Chemical Changes of Bases
Problem
• Hypoxanthine is an oxidized (deaminated)
derivative of adenine. As the nucleoside
inosine, it can base-pair with both cytidine
and adenosine. Draw the structures of these
base-pairs.
O
H N
N
I
N
N
H
OH
O
OH
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H
O
N H
O
N
I
H N
N
HO
N
N
N
O
H
O
OH
OH
HO
H
N H
N
O
N
N
N
H N I
N
HO
O
N
N
OH
O
H
OH
OH
H
Deamination of A yields
Hyp, which leads to A
à G mutations
C N
O
HO
A
D
O
N H
N
D
A
I
H N
N
N
N
O
H
O
OH
Normally, this A/D
OH pattern is in the
HO
H
N H
N
O
HO
Deamination of A yields
OH
Hyp, which leads to A
à T mutations
N
A N
A
pyrimidine pattern
(T), or the first part
of the G pattern
(A/D/D).
A
D
O
N
D
N
H N I
N
N
H
OH
O
OH
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Stability of the Polymers: Nucleic Acids
Acid/base treatment of DNA
In Base: deprotonation at G (N1) & U (N3) destabilizes the
glycosidic bond, which leads to AP-sites.
In Acid: protonates at A (N1), C (N3), & G (N7). For C & G,
this also destabilizes the glycosidic bond, which leads to APsites
AP-sites can lead to cleavage of the phosphodiester bond
Acid/base treatment of RNA
Similar generation of AP sites, except importantly in Base:
complete cleavage of the phosphodiester bonds!
Stability of the Polymers: Nucleic Acids
Base treatment of RNA
Base
Base
Base
OH–
H2O
H+
Base
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Stability of the Polymers: Nucleic Acids
Base treatment of RNA
Base
Base
Base
OH–
H2O
H+
Base
Stability of the Polymers: Nucleic Acids
DNA Denaturation
dsDNA ⇌ ssDNA
Acid/base puts charges on
bases, which causes
internal repulsions and
breaks up the double helix
This denaturation can also
be accomplished using
heat.
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Stability of the Polymers: Nucleic Acids
DNA Denaturation
UV Spectrum: Native vs. Denatured DNA
This denaturation
causes a
hyperchromic
shift.
40%
increase
This makes a
nice “observable”
(A260) and
“perturbable”
(heat).
Stability of the Polymers: Nucleic Acids
DNA Denaturation
DNA Melting Curve
This Tm value is
dependent on a
number of
parameters.
Like protein denaturation,
this is a cooperative
process.
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Stability of the Polymers: Nucleic Acids
DNA Denaturation
This Tm value is dependent on:
• [salt]
• solvents (urea, formamide, guanidine salts)
• G:C content of sequence
1x SSC (Salt-Sodium Citrate)
Stability of the Polymers: Nucleic Acids
DNA Renaturation
ssDNA ⇌ dsDNA
•Also called re-annealing or hybridization
•Depends on conditions (temp, [salt], solvent) that are
maintained BELOW the Tm value.
•In addition, the proper formation of the complete,
pristine, double helix (completely double-stranded)
requires the proper amount of TIME and
CONCENTRATION of nucleic acid.
•Plots of this are called Cot curves, which are much like
Tm curves.
•Cot values are dependent on the complexity of the
sequence.
•Not enough time, you get scrambled structures
Partially
•Given enough time, very specific annealing occurs….
Renatured DNA
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Stability of the Polymers: Nucleic Acids
DNA Renaturation
EXAMPLE:
Karyotyping
Fluorescence in situ hybridization (FISH)
This FISH probe (a fluorescent DNA) found its
FISH using fluorescence probe for gene on complementary sequence among
3,000,000,000 bp in each cell!
chromosome 21.
Stability of the Polymers: Nucleic Acids
DNA Renaturation
EXAMPLE:
Karyotyping
Fluorescence in situ hybridization (FISH)
This FISH probe (a fluorescent DNA) found its
FISH using fluorescence probe for gene on complementary sequence among
3,000,000,000 bp in each cell!
chromosome 21.
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Stability of the Polymers: What is
responsible for STABILITY of dsDNA?
C:C nearly as
stable as A:U
Hoogstein bp is very stable
G:G more
stable than
A:U and as
much as G:C
Stability of the Polymers: What is
responsible for STABILITY of dsDNA
What forces operate? If it’s not the H-bonds, then what is it?
• Ionic/electrostatics (salt-bridges)?
It’s a poly-anion; so charges actually de-stabilize
• Hydrophobic?
Unlike proteins, where this is the driving force, experiments show that the ssDNA ⇌
dsDNA reaction is enthalpy driven process; ∴ bonds
• van der Waals?
Yes! This is the most important.
As uniform bp come together due to
complementarity, the planer bases “stack” on each other and are close enough (<2 Å) to generate
induced dipoles.
Once started, it “zips” together as long as there are complementary bp being formed.
Stacking energy = stability
H-bonds in complementary bp = specificity
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Stability of the Polymers: What is
responsible for STABILITY of dsDNA
So, if its stacking energy, why are G:C rich
sequences more stable than A:T rich
sequences?
Stacking Energies in B-DNA
Adenine ring stacking
Stability of the Polymers: Biochemical
•Enzymes that catalyze the hydrolysis of the phosphodiester
bonds:
•These enzymes are called “Nucleases”
•Like proteases, if they cleave in the middle, they are
called endonucleases (e.g., restriction endonucleases)
•If they cleave at the ends, they are called exonucleases
Exonucleases can be specific for either 5’-ends or 3’-ends, or
either double-stranded or single-stranded nucleic acids (e.g., S1
nuclease)
•Also specificity for either DNA (DNases) or RNA (RNAses)
•RNAases are very stable, DNAases require Mg+2
cofactor
•Can be inhibited by DEPC or EDTA, respectively
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Nucleotides & Nucleic Acids: preliminary concepts and
checkpoints
Key Concepts
• The nitrogenous bases of nucleotides include two types of purines and
three types of pyrimidines.
• A nucleotide consists of a nitrogenous base, a ribose or deoxyribose sugar,
and one or more phosphate groups.
• DNA contains adenine, guanine, cytosine, and thymine
deoxyribonucleotides, whereas RNA contains adenine, guanine, cytosine,
and uracil ribonucleotides.
• Phosphodiester bonds link nucleotide residues in DNA and RNA.
• In the DNA double helix, two antiparallel polynucleotide strands wind
around each other, interacting via hydrogen bonding between bases in
opposite strands.
• RNA molecules are usually single-stranded and may form intramolecular
base pairs.
• DNA carries genetic information in the form of its sequence of nucleotides.
• The nucleotide sequence of DNA is transcribed into the nucleotide
sequence of messenger RNA, which is then translated into a protein, a
sequence of amino acids.
Nucleotides & Nucleic Acids: preliminary concepts and
checkpoints
Checkpoint
• Identify the purines and pyrimidines commonly found in nucleic acids.
• Practice drawing the structures of adenine, adenosine, and adenylate
• Describe the chemical differences between a ribonucleoside triphosphate
and a deoxyribonucleoside monophosphate.
• Using Fig. 3-3a as a guide, draw the complete structure of a nucleoside
triphosphate before and after it becomes incorporated into a polynucleotide
chain. Draw the structure that would result if the newly formed
phosphodiester bond were hydrolyzed.
• Explain the structural basis for Chargaff’s rules.
• Using a three-dimensional computer model of the DNA molecule, identify
each of the following structural features: the 3‘ and 5’ end of each strand, the
atoms that make up the sugar–phosphate backbone, the major and minor
grooves, the bases in several base pairs, and the atoms that participate in
hydrogen bonding in A·T and G·C base pairs.
• List the structural differences between DNA and RNA.
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