1 O O P O O O N HO N N O H H O H H N H H H O O P O O O N HO N O O H deoxycytidine C deoxyuridine U Why Does DNA Use T Instead of U? Problem: deaminated C is identical to U (and pairs with A) deamination G–C G–U G DNA repair machinery removes all Us from DNA DNA repair G–C • DNA uses T instead of U to avoid mutations arising from damaged (deaminated) C, which is identical to U ~100 per day per cell Lectures 3-5: Nucleic acids & the chemical requirements for replicating information 1. The primary biological roles of nucleic acids 2. The molecular components of DNA and RNA a. The primary structure of deoxyribonucleic acid b. The phosphate group in DNA; equilibrium, acidity, and protonation states c. The sugar group in DNA; strand orientation and macromolecular chirality d. The bases of DNA e. The primary structure of ribonucleic acid f. Why does DNA use deoxyribose? Why T? 3. The factors behind DNA base pairing a. DNA hybridization as an equilibrium b. The role of hydrogen bonding c. The role of the hydrophobic effect and base stacking 4. The molecular basis of DNA replication a. DNA replication; chemical reactions, substrates, and products b. The role of DNA polymerase: faster and more accurate DNA replication c. The polymerase chain reaction (PCR) and its impact on the life sciences
Transcript
1
OOPOO
O
N
HO
N
N
O
H H
O
H H
N
H
HH
OOPOO
O
N
HO
N
O
O
H
deoxycytidineC
deoxyuridineU
Why Does DNA Use T Instead of U?
Problem: deaminated Cis identical to U (andpairs with A)
deaminationG–C G–U G
DNA repairmachinery
removes all Usfrom DNA DNA repair
G–C
• DNA uses T instead of U to avoid mutations arising fromdamaged (deaminated) C, which is identical to U
~100 perday per cell
Lectures 3-5: Nucleic acids & the chemical requirements for replicating information
1. The primary biological roles of nucleic acids
2. The molecular components of DNA and RNA
a. The primary structure of deoxyribonucleic acid
b. The phosphate group in DNA; equilibrium, acidity, and protonation states
c. The sugar group in DNA; strand orientation and macromolecular chirality
d. The bases of DNA
e. The primary structure of ribonucleic acid
f. Why does DNA use deoxyribose? Why T?
3. The factors behind DNA base pairing
a. DNA hybridization as an equilibrium
b. The role of hydrogen bonding
c. The role of the hydrophobic effect and base stacking
4. The molecular basis of DNA replication
a. DNA replication; chemical reactions, substrates, and products
b. The role of DNA polymerase: faster and more accurate DNA replication
c. The polymerase chain reaction (PCR) and its impact on the life sciences
2
DNA Replication Relies on Base Pairing
A - TT - AG - CG - CT - AC - GA - TG - C
A - TT - AG - CG - CT - AC - GA - TG - C
A - TT - AG - CG - CT - AC - GA - TG - C
DNA replication
+
• Under typical conditions, Keq >> 1 and there are many moredouble-stranded molecules than single-stranded molecules
• What causes DNA hybridization to be favorable?
A B C
Keq = [A][B][C]
DNA Hybridization Equilibrium
>> 1 (e.g., 10,000 M-1)
3
Double-stranded:4 hydrogen bonds
+ +
Hydrogen Bonding: Matched Base Pairs
• The same # of H-bonds are possible on both sides, althoughscientists continue to debate how these H-bonds differ in strength
H
O H O
H
H
H
OHO
H
H
N
N
N
N
N
H
H
H
O
H
H
O H
N
N
N
N
N
H
H
N
N
CH3O
O
H
N
N
CH3O
O
H
H
O
H
OH
H
Single-stranded:4 hydrogen bonds
Within a double helix
Hydrogenbonding
alone maynot stronglyfavor either
side
• Hydrogen bonding alone may not induce DNA hybridization,but the loss of hydrogen bonds disfavors mismatched pairing
N
N
N
O
H
H
N
N
N
N
N
H
H
+ +
Hydrogen Bonding: Mismatched Base Pairs
Hydrogenbonding
alone favorsthe unpaired
side formismatched
basesH
O H O
H
H
H
OHO
H
H
N
N
N
N
N
H
H
H
O
H
H
O H
N
N
N
O
H
O H
O
H
H
H
H
Double-stranded:2 hydrogen bonds
Single-stranded:4 hydrogen bonds
Within a double helix; noroom for water molecules
here
Keq = ~ 0.01
4
The Hydrophobic Effect
OH
H
O
H
H
O
H H
OH
H
O
H
H O
HH
OH
HO
H
H
O
HH
O
H
H
O
H
H
CH2
H2C
CH2
H2C
CH2
H2C
H3C
CH3O
H
HO
H
HOHH
OH
H
O
H
HO
H
H
O H
H
O
H
H
O
HHO
HH
O
H
H
H2C
CH2
H2C
CH2
H2C
CH2
CH3
H3C
Water forms an ordered “lattice”around hydrophobic (oily) surfaces
OH
H
OH H
O
H
H
OH
H
O
H
H
O
H HO
H
CH2H2C
CH2H2C
CH2H2C
H3C
CH3O
H
H
OHH
O
H
H
O H
H
O
H
H
O
HHO
H
H2C
CH2
H2C
CH2
H2C
CH2
CH3
H3C
H
H
• Ordered waters are disfavored because they are capable of lessmotion (they have less entropy)
• Fewer water molecules are ordered when hydrophobic groups aregathered together
OH
H
O
HH
O
H
H
O
H
H
OH
H
O
H
H
O
H
H
O
H
H
disorderedwater
+
orderedwater
N
N
N
N
NH H
HN
N
N
N
NH H
H
The Nucleic Acid Bases haveHydrophobic Surfaces
Top and bottomsurfaces arehydrophobic
Rotate 90o
• Water exposure of thesehydrophobic surfaces isminimized by stacking thebases
• In double-stranded DNA,the bases are largelystacked
N
N
N
N
NH H
H
N
N
N
N
NH H
H
Not accessibleto water
Bases stacked as indouble-stranded DNA
5
• DNA hybridization minimizeswater-exposed hydrophobicsurfaces
DNA Hybridization is Largely Drivenby the Hydrophobic Effect
No room for water molecules!
Lectures 3-5: Nucleic acids & the chemical requirements for replicating information
1. The primary biological roles of nucleic acids
2. The molecular components of DNA and RNA
a. The primary structure of deoxyribonucleic acid
b. The phosphate group in DNA; equilibrium, acidity, and protonation states
c. The sugar group in DNA; strand orientation and macromolecular chirality
d. The bases of DNA
e. The primary structure of ribonucleic acid
f. Why does DNA use deoxyribose? Why T?
3. The factors behind DNA base pairing
a. DNA hybridization as an equilibrium
b. The role of hydrogen bonding
c. The role of the hydrophobic effect and base stacking
4. The molecular basis of DNA replication
a. DNA replication; chemical reactions, substrates, and products
b. The role of DNA polymerase: faster and more accurate DNA replication
c. The polymerase chain reaction (PCR) and its impact on the life sciences
6
How Does DNA Replicate?
Deoxynucleotidetriphosphates
Template
Primer
Extendedprimer
OO N
N
N
N
O
HO
N
H
H
HPOO
O
PO
POO O
O O
OO N
HO
NH3C
O
O
H
POO
O
PO
POO O
O O
OOPOO
O
N
N
N
N
N
HO
H H
PO
POO O
O O
OO N
HO
N
N
O
H H
POO
O
PO
POO O
O O
dATP dCTP
dTTPdGTP
Polymerization
A
T
G
G - ?
T - A
C - G
A - T
G - C
Base Pairing Determines SelectivityDuring DNA Polymerization
O
OPO
O
O
NOH
POPO
O
O
O
O
N
O
O CH3
H
O
OPO
O
O
N
N
N
N
O
OH
POPO
O
O
O
O
H
NH
HO
OPO
O
O
N
N
N
N
N
OH
H
H
POPO
O
O
O
O
O
OPO
O
O
NOH
POPO
O
O
O
O
N
O
NH
H
template primer
dTTP
dATP dGTP
dCTP
pyrophosphateOPOPO
O
O
O
O
Extended primer withnew 3’ OH group
• The nucleotidecapable of forminga base pair with thetemplate is addedto the primer
O
O
PO
O
O
N
N
N
N
O
O N
H
H
H
O
O
P
O
O
N
O
N
O
O
H
H3C
O
O
P O
O
O
N
N
N
N
N
OH
H
H
O
OPO
O
O
OH
POPO
O
O
O
O
Base
:
O
O
PO
O
O
N
N
N
N
O
O N
H
H
H
O
O
P
O
O
N
O
N
O
O
H
H3C
O
O
P O
O
O
N
N
N
N
N
O
H
HO
O P
OO
NOH
N
O
NH
H
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DNA Polymerization, South Park-Style
A T G GT - AC - GA - TG - C
----3’
5’
5’® Comedy Central (don’t sue me, please)
O
O
PO
O
O
N
N
N
N
O
O N
H
H
H
O
O
P
O
O
N
O
N
O
O
H
H3C
O
O
P O
O
O
N
N
N
N
N
OH
H
H
DNA Polymerization in Action I
A T G GT - AC - GA - TG - C
template primer
5’
3’ 5’
----3’
5’
5’
dATPdCTP
dGTPdTTP
3’
8
OO
PO
O
O
NOH
POPO
O
O
O
O
N
O
NH
H
O
O
PO
O
O
N
N
N
N
O
O N
H
H
H
O
O
P
O
O
N
O
N
O
O
H
H3C
O
O
P O
O
O
N
N
N
N
N
OH
H
H
DNA Polymerization in Action II
A T G GT - AC - GA - TG - C
template primer
5’
3’ 5’
----3’
5’
5’
(dCTP)
3’
- CTP
dATP
dCTPdGTP
dTTP
O
O
PO
O
O
N
N
N
N
O
O N
H
H
H
O
O
P
O
O
N
O
N
O
O
H
H3C
O
O
P O
O
O
N
N
N
N
N
O
H
HO
O
P
O
O
N OH
N
O
NH
H
DNA Polymerization in Action III
A T G GT - AC - GA - TG - C
template primer
5’
3’ 5’
----3’
5’
5’
new 3’ OH groupof growing strand
- C
OP
OPO
O
O
O
O
pyrophosphatedATP
dCTPdGTP
dTTP
9
O
O
P
O
O
N
N
N
N
O
O N
H
H
H
O
O
P
O
O
N
O
N
O
O
H
H3C
O
O
P O
O
O
N
N
N
N
N
O
H
HO
O
P
O
O
N OH
N
O
NH
H
O
O
PO
O
O
N
N
N
N
O
O N
H
H
H
DNA Polymerization in Action IV
A T G GT - AC - GA - TG - C
template primer
5’
3’ 5’
----3’
5’
5’
- C
dATPdCTP
dGTP
dTTP
OO
PO
O
O
NOH
POPO
O
O
O
O
N
O
NH
H
O
O
P
O
O
N
N
N
N
O
O N
H
H
H
O
O
P
O
O
N
O
N
O
O
H
H3C
O
O
P O
O
O
N
N
N
N
N
O
H
HO
O
P
O
O
N OH
N
O
NH
H
O
O
PO
O
O
N
N
N
N
O
O N
H
H
H
DNA Polymerization in Action V
A T G GT - AC - GA - TG - C
template primer
5’
3’ 5’
----3’
5’
5’
- C
- CTP
dATPdCTP
dGTPdTTP
(dCTP)
10
DNA Polymerization in Action VI
A T G GT - AC - GA - TG - C
template primer
5’
3’ 5’
----3’
5’
5’
- C
- C
dATPdCTP
dGTPdTTP
O
O
P
O
O
N
N
N
N
O
O N
H
H
H
O
O
P
O
O
N
O
N
O
O
H
H3C
O
O
P O
O
O
N
N
N
N
N
O
H
H
OO
P
O
O
N
ON
O
NH
H
O
O
PO
O
O
N
N
N
N
O
O N
H
H
H
O
O
PO
O
NOH
N
O
NH
H
Note that thenew strand isgrowing in the5’ to 3’ direction
DNA Polymerase Accelerates Replication
OPOPO
O
O
O
O
OPOPO
O
O
O
O
A
T
G
G
T - A
C - G
A - T
G - C
A
T
G
G
T - A
C - G
A - T
G - C
A
T
G
G - C
T - A
C - G
A - T
G - C
A
T
G
G - C
T - A
C - G
A - T
G - C
dCTP +
dCTP +Fast
(~50 bases addedper second)
Very slow(no observable
reaction)
DNA polymerase
11
DNA Polymerization in Cells, CG-Style
A T G GT - AC - GA - TG - C
----3’
5’
5’
• DNA polymerization in cells requires DNApolymerase plus several other proteins tounwind double-stranded DNA and to performseveral other essential tasks)
Animation by Drew Berry, used with permission
DNA Replication is Extremely Accurate
Selectivity based onhydrogen bonding alone:
With all cellular machinery:
With DNA polymerase:
Method Error Rate
~1 in 100
~1 in 100,000,000
~1 in 10,000,000,000
12
The Polymerase Chain Reaction (PCR)
PrimersPolymerase
OO N
N
N
N
O
HO
N
H
H
HPOO
O
PO
POO O
O O
OO N
HO
NH3C
O
O
H
POO
O
PO
POO O
O O
OOPOO
O
N
N
N
N
N
HO
H H
PO
POO O
O O
OO N
HO
N
N
O
H H
POO
O
PO
POO O
O O
Four dNTPs
Melt template(heat)
Hybridizeprimers(cool)
Extendprimers
Repeat
5’3’
3’5’5’3’
3’5’
5’ 3’
3’ 5’
The PCR Cycle
13
PCR Exponentially Amplifies DNA
One DNA template molecule after 25 PCR rounds gives225 = 33,554,432 molecules!
Thermus aquaticus (Taq) DNA Polymerase
• Taq DNA polymerase: operates at 74 °C (extensiontemperature), tolerant of 95 °C (melting temperature)
• Thermostable polymerases (and the diversity of life onearth) made PCR practical
14
PCR Applications: Molecular BiologyA gene of interest can be amplifiedand inserted into a model organism
PCR using primers thatmatch the ends of the gene
Introduce geneinto bacteria
Bacteria use geneto make protein
Isolate protein,study its function
PCR with mutation-containing primer
Reassemblemutant gene
Make mutant protein; compareproperties to wild-type protein
mutantprotein
wild-typeprotein
A specific mutation in a protein-encoding gene can be introduced
genomic DNA
Make mutant protein
vs.
PCR Applications: HIV Detection• Because PCR can amplify extremely small quantities of DNA,
it can be used as a sensitive method of pathogen detection
Blood sample
Isolate HIV genome(RNA)
Convert to DNAPCR No PCR product
indicates nodetectable HIV levels
PCRamplificationindicatespresence of HIV
• Sensitivity limit = ~10-100 copies of HIV genome
15
Key Points: How DNA Meets theRequirements for the Blueprint of Life
• Resist degradation: negatively charged phosphates; no 2’ OH(equilibrium, acidity, Ka, pKa, pH, Henderson-Hasselbalch)
• Be recognized by cellular machinery: phosphate groups andbases (ionic bonds and hydrogen bonds)
• Contain multiple possible structures (bits) at each position: fourpossible bases per nucleotide
• Possess redundancy for error correction and replication: basepairing (hydrogen bonding, hydrophobic effect)
• Knowledge of DNA replication lead to PCR, a key invention• Understanding the chemistry of DNA is crucial to the life sciences
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