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Introduction to DNAIntroduction to DNALecture notes edited by John Reif from PPT lectures by:Lecture notes edited by John Reif from PPT lectures by:
Image from
http://zen-haven.dkhttp://zen-haven.dk
Natalia Tretyakova, College of Pharmacy, U. of Minnesota
Richard Lavery, Institut de Biologie Physico-Chimique, Paris
• DNA
• Double helix
• Stores genetic code as a linear sequence of bases
• ≈ 20 Å in diameter
• Human genome ≈ 3.3 x 109 bp
• ≈ 25,000 genes
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
DNA Size Scale
Biological length scale
Chemical bond 1 Å (10-10 m)
Amino acid 10 Å (10-9 m)
Globular protein 100 Å (10-8 m)
Virus 1000 Å (10-7 m)
Cell nucleus 1 m (10-6 m)
Bacterial cell 5 m (10-5 m)
Chromosome DNA 10 cm (10-1 m)
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
DNA BASES
Nucleoside
Nucleotide
OH ribose
H deoxyribose
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
The Building Blocks of DNA
Nucleotides are linked by phosphodiester bonds
Strand has a direction (5'3')
DNA is negatively charged on phosphate backbone.
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
Base families
Purine (Pur / R) Pyrimidine (Pyr / Y)
C2
N1
C5C6N7
C4
C8
N9
N3
N1
C4
N3
C2
C5
C6
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
DNA and RNA nucleobases
NN
NNH
NH2
NNH
NNH
O
NH2
N
NH
NH2
O
H3C
NH
NH
O
O
Guanine (G)Adenine (A)
Thymine (T)Cytosine (C)
NH
NH
O
O
Uracil (U)
NN
NNH
Purine
N
NH
Pyrimidine
1
2
3
4
5
6
3
2
16
4
57
8
9
•(DNA only) •(RNA only)
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
Purine BasesThe 9 atoms that make up the fused rings (5 carbon, 4 nitrogen) are numbered 1-9.
All ring atoms lie in the same plane.
Richard B. Hallick
Introductory Course in Biology or Biochemistry
Purine Nucleotides
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
Pyrimidine BasesAll pyrimidine ring atoms lie in the same plane.
Richard B. Hallick
Introductory Course in Biology or Biochemistry
Pyrimidine Nucleotides
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
•nucleobase •(Deoxy)•nucleoside
•5’-mononucleotide
•Adenine (A)
•Guanine (G)
•Thymine (T)
•Cytosine (C)
•Uracil (U)
•2’-Deoxyadenosine (dA)•2’- Deoxyguanosine (dG)•2’- Deoxythymidine •(dT)•2’- Deoxycytidine •(dC)•Uridine (U)
•Deoxyadenosine 5’-monophosphate •(5’-dAMP)•Deoxyguanosine 5’-monophosphate •(5’-dGMP)•Deoxythymidine 5’-monophosphate •(5’-dTMP)•Deoxycytidine 5’-monophosphate •(5’-dCMP)•Uridine 5’-monophosphate (5’-UMP)
• • • Nomenclature of nucleobases, nucleosides,
and mononucleotides
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
Structural differences between DNA and RNA
H3CNH
NH
O
O
Thymine (T)
NH
NH
O
O
Uracil (U)
•DNA •RNA
O
H
HHH
CH2
HO
HOBase
2'-deoxyribose
O
OH
HH
CH2
HO
HOBase
ribose
H
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
Deoxyribose Sugar
The hydroxyl groups on the 5'- and 3'- carbons link to the phosphate groups to form the DNA backbone.
Richard B. Hallick
Introductory Course in Biology or Biochemistry
Richard B. Hallick
Introductory Course in Biology or Biochemistry
Nucleosides•A nucleotide is a nucleoside with one or more phosphate groups covalently attached to the 3'- and/or 5'-hydroxyl group(s).
Preferred conformations of nucleobases and sugars in DNA and RNA
HO
O
OH
N
N
NH2
O
Anti conformation
HO
O
OH
N
N
NH2
O
Syn conformation
HO
OH (OH)
HO
BASEHO
O
H (OH)
HOBASE
2' endo (B-DNA)
1'
3' endo (RNA)
3'
1'3'
2' 5'5'
•7.0 A
• •5.9 A
•Sugar puckers:
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
Nucleosides Must Be Converted to5’-Triphosphates to be Part of DNA and
RNA
HOO
OH
OO
OH
PHO
HO
O
OO
OH
P
O
P
OHO
HOO
OH
Base Base
BaseO
O
OH
P
O
P
O
O
OHBase
OH
OP
OHO
HO
Kinase
Kinase
Kinase
Monophosphate
DiphosphateTriphosphate
ATP
ATP
ATP
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
DNA BASE PAIRING
Watson-Crick base pairs
Thymine -Adenine Cytosine -Guanine
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
Richard B. Hallick
Introductory Course in Biology or Biochemistry
A-T and G-C Base Pairing
Hydrogen bond donors and acceptors on each edge of a base pair
NH
N
N O
NH2
N NN
H2N
O
HN
N
O
O
NN
N
N NH2
G•C A•T
NO
H
OH
N H
O
NN
O
Major groove
Minor groove
To deo
xyrib
ose
To deoxyribose
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
Purine always binds with a Pyrimidine
Base pair dimensionsRichard Lavery
Institut de Biologie Physico-Chimique, Paris
DNA/RNA chemical structure
DNA : A ,T,G,C + deoxyriboseRNA : A,U,G,C + ribose
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
DNA BACKBONE STRUCTURE
Richard B. Hallick
Introductory Course in Biology or Biochemistry
Backbone structure:• Alternating backbone of deoxyribose and phosphodiester groups• Chain has a direction (known as polarity), 5'- to 3'- from top to bottom• Oxygens (red atoms) of phosphates are polar and negatively charged• Bases extend away from chain, and stack atop each other• Bases are hydrophobic
Helix Axis View:
OnScreen DNA Model app
B-DNA STRUCTURE
Video of DNA Helix Structure:
http://www.youtube.com/watch?v=ZGHkHMoyC5I
Contains material from:
Alberts, Bray, Hopkin, Johnson, Lewis, Raff, Roberts, Walter, Essential Cell Biology, Second Edition, Garland Science Publishing, 2004
34 Å
3.4 Å
20 Å
MinorGroove
MajorGroove
GC
CG
AT
TA
CG
GCAT
TA
TA
AT
GCCG
GC
Strands areantiparallel
CGCGTTGACAACTGCAGAATC
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
B-DNA Structure
Richard B. Hallick
Introductory Course in Biology or Biochemistry
Features of the B-DNA Double Helix•Two DNA strands form a helical spiral, winding around a helix axis in a right-handed spiral •The two polynucleotide chains run in opposite directions •The sugar-phosphate backbones of the two DNA strands wind around the helix axis like the railing of a sprial staircase •The bases of the individual nucleotides are on the inside of the helix, stacked on top of each other like the steps of a spiral staircase.
B-DNA (axial view)Richard Lavery
Institut de Biologie Physico-Chimique, Paris
B-DNA (lateral view)
R.H. helix
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
•Base stacking: an axial view of B-DNA
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
PI Bonds – (Mechanism of PI Base Stacking)
Forces stabilizing DNA double helix
1. Hydrogen bonding (2-3 kcal/mol per base pair)
2. Stacking (hydrophobic) interactions (4-15 kcal/mol per base pair)
3. Electrostatic forces.
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
Comparison to other bonds1. Covalent Bond Energies:
1. C-C 85 kcal/mol2. C-O 87 kcal/mol
•right handed helix
• planes of bases are nearly
•perpendicular to the helix axis.
••Sugars are in the 2’ endo conformation.
••Bases are the anti conformation.
••Bases have a helical twist of 34.6º (10.4 bases per helix turn)
• Helical pitch = 34 A
•B-DNA
• 3.4 A rise between base pairs
•Wide and deep
•Narrow and deep
HO
O
OH
N
N
NH2
O
HO
OH (OH)
HO
BASE
1'3'
2'5'
•7.0 A
• helical axis passes through•base pairs
•23.7 A
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
•DNA can deviate from the ideal Watson-Crick structure
• Helical twist ranges from 28 to 42°
• Propeller twisting 10 to 20°
•Base pair roll
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
DNA grooves
MINOR
MAJOR
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
Major groove and Minor groove of DNA
Major groove
Minor groove
•NH
•N
•N•O
•NH•2
•N •N•N
•H•2•N
•O•C-1’•C-1’
•HN
•N
•O
•O
•N•N
•N
•N •NH•2
•C-1’
•C-1’
Major groove
Minor groove
Base Base•To deoxyribose-C1’ •C1’ -To deoxyribose
•Hypothetical situation: the two grooves would have similar size if dR residues •were attached at 180° to each other
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
•Major and minor groove of the double helix
•Wide and deep
•Narrow and deep
Major groove
•NH
•N
•N •O
•NH•2
•N •N•N
•H•2•N
•O
Minor grooveTo deo
xyrib
ose •C-1’
•C-1’
•HN
•N
•O
•O
•N•N
•N
•N •NH•2
•C-1’
•C-1’
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
•B-type duplex is not possible for RNA
•steric “clash”
O
OH
HH
CH2
HO
HOBase
ribose
H
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
A-DNA STRUCTURE
A and B DNA allomorphs
B A
Hydration
Antiparallel strands
5’5’3’
3’
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
De-hydration
A-DNA (longitudinal view)Richard Lavery
Institut de Biologie Physico-Chimique, Paris
A-DNA (lateral view)
R.H. helix
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
•A-form helix: dehydrated DNA; RNA-DNA hybrids
•Top View
•Right handed helix
• planes of bases are tilted
•20 ° relative the helix axis.
• 2.3 A rise between base pairs
•
••Sugars are in the 3’ endo conformation.
••Bases are the anti conformation.
••11 bases per helix turn
• Helical pitch = 25.3 A
•25.5 A
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
The sugar puckering in A-DNA is 3’-endo
O
OH (OH)
O
BASEO
O
H (OH)
OBASE
2' endo (3' exo) B-DNA
1'
3' endo (A-DNA)
3'
1'3'
2'
5'
5'
2'
•7.0 A
•5.9 A•
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
A-DNA has a shallow minor groove and a deep
major groove
N
NH
N
N
O
NH2
NN
H2N
O
To deo
xyrib
ose To deoxyribose
Major groove
Minor groove
•B-DNA
N
NH
N
N
O
NH2
NN
H2N
O
To deo
xyrib
ose To deoxyribose
Major groove
Minor groove
•Helix axis
•A-DNA
•• ••
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
Z-DNA STRUCTURE
Z-DNA (longitudinal view)Richard Lavery
Institut de Biologie Physico-Chimique, Paris
Z-DNA (lateral view)
L.H. helix
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
Base pairs are rotated in Z-DNARichard Lavery
Institut de Biologie Physico-Chimique, Paris
•Z-form double helix: polynucleotides of alternating purines and pyrimidines (GCGCGCGC) at high salt
•Left handed helix
•
•• Backbone zig-zags because sugar puckers alternate between 2’ endo pyrimidines and 3’ endo (purines)
•• Bases alternate between anti (pyrimidines) and syn conformation (purines).
••12 bases per helix turn
• Helical pitch = 45.6 A
• planes of the bases are
•tilted 9° relative the helix
•axis.
•• Flat major groove
•• Narrow and deep minor groove
•18.4 A
• 3.8 A rise between base pairs
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
Sugar and base conformations in Z-DNA alternate:
N
N
NH2
ON
HN
NN
O
H2NHO
OH
HO
HO
O
H
HO1' 3'
1'3'
2'
5'
5'
GC
•5’-GCGCGCGCGCGCG•3’-CGCGCGCGCGCGC
•C: sugar is 2’-endo, base is anti•G: sugar is 3’-endo, base is syn
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
Comparing A, B and Z-DNA
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
• Biological relevance of the minor types of DNA secondary structure
•Although the majority of chromosomal DNA is in B-form, •some regions assume A- or Z-like structure
• Runs of multiple Gs are A-like
•The upstream sequences of some genes contain •5-methylcytosine = Z-like duplex
N
NH
NH2
O
5-methylcytosine (5-Me-C)
H3C
• RNA-DNA hybrids and ds RNA have an A-type structure
• Structural variations play a role in DNA-protein interactions
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
Backbone Dihedrals
Backbone dihedrals - I
0
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
+60°+10°
Dihedral angle definition
Staggered Eclipsed
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
Favoured conformations
gauche +
trans
gauche -
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
Backbone dihedrals - II
: O3’ – P – O5’ – C5’ g-
: P – O5’ – C5’ – C4’ t
: O5’ – C5’ – C4’ – C3’ g+
: C5’ – C4’ – C3’ – O3’ g+
: C4’ – C3’ – O3’ – P t
: C3’ – O3’ – P – O5’ g-(Y) : O4’ – C1’ – N1 – C2 g-
(R) : O4’ – C1’ – N9 – C4
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
syn-anti glycosidic conformationsRichard Lavery
Institut de Biologie Physico-Chimique, Paris
Sugar ring puckering
C5’
ENDO
EXO
Base
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
Sugar pucker described as
pseudorotation
North : C3’-endo
East : O4’-endo
South : C3’-endo
"2 B or not 2 B ...." W. Shakespeare 1601
Pseudorotation Equations
Altona et al. J. Am. Chem. Soc. 94, 1972, 8205
0
2
13
4
Basetan P = (4 - 1) - (3 - 0)
22 (Sin 36° + Sin72°)
Amp = 2 / Cos P
Preferred sugar puckersRichard Lavery
Institut de Biologie Physico-Chimique, Paris
Sugar pucker and P-P distanceRichard Lavery
Institut de Biologie Physico-Chimique, Paris
UNUSUAL DNA STRUCTURES
Alternative base pairs
Watson-Crick
Reversed Watson-Crick
Hoogsteen Reversed Hoogsteen
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
Watson-Crick + Hoogsteen = Base triplet
- note C(N3) protonation
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
Triple helix DNARichard Lavery
Institut de Biologie Physico-Chimique, Paris
Guanine Hoogsteen pairing Base tetraplexRichard Lavery
Institut de Biologie Physico-Chimique, Paris
Robert E Johnson et. al
University of Texas Medical Branch
Watson Crick vs Hoogsteen Hydrogen Bonding.
(inset, G-C bonding also shown)
Quadruplex DNARichard Lavery
Institut de Biologie Physico-Chimique, Paris
Inverted repeat can lead to loop formationRichard Lavery
Institut de Biologie Physico-Chimique, Paris
DNA cruciform
Holliday junction
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
PNA versus DNARichard Lavery
Institut de Biologie Physico-Chimique, Paris
Peptide Nucleic acid(PNA)
Achiral, peptide-like backbone
Backbone is uncharged High thermal stability
High-specificity hybridization with DNA
Resistant to enzymatic degradation
Can displace DNA strand of duplex
Pyrimidine PNA strands can form 2:1 triplexes with ssDNA
Biotechnological applications
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
Parallel-stranded DNARichard Lavery
Institut de Biologie Physico-Chimique, Paris
I-DNA: intercalated parallel-stranded duplexes Richard Lavery
Institut de Biologie Physico-Chimique, Paris
and nucleotide anomers Richard Lavery
Institut de Biologie Physico-Chimique, Paris
H OH is not the only change in passing from DNA to RNA .... Richard Lavery
Institut de Biologie Physico-Chimique, Paris
Biophysical properties of DNA
Biophysical properties of DNA
T, C70 80 90 100
A260
TM
• Facile denaturation (melting) and re-association of the duplex • are important for DNA’s biological functions.• In the laboratory, melting can be induced by heating.
• Hybridization techniques are based on the affinity of complementary
• DNA strands for each other.
• Duplex stability is affected by DNA length, % GC base pairs, ionic strength, the presence of organic solvents, pH
• Negative charge – can be separated by gel electrophoresis
•T°
•Single strands
•duplex
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
•Separation of DNA fragments by PAGE
• DNA strands are negatively charged .• Migrate towards the (+) electrode (anode)• Migration time ~ ln ( number of base pairs)
Books on DNA
Principles of Nucleic Acid Structure, W. Saenger, 1984 Springer-Verlag
Nucleic Acid Structure, Ed. S. Neidle, 1999 Oxford University Press
DNA Structure and Function, R.R. Sinden, 1994 Academic Press
Biochemistry, D. Voet and J.G. Voet, 1998 DeBoeck
The Eighth Day of Creation, H.F. Judson, 1996 Cold Spring Harbour Press
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
HISTORY of DNA
History of DNA
1865 Gregor Mendel publishes his work on plant breeding with the notionof "genes" carrying transmissible characteristics
1869 "Nuclein" is isolated by Johann Friedrich Miescher à Tübingen
in the laboratory of Hoppe-Seyler 1892 Meischer writes to his uncle "large biological molecules composed
of small repeated chemical pieces could express a rich language inthe same way as the letters of our alphabet"
1920 Recognition of the chemical difference between DNA and RNA
Phoebus Levene proposes the "tetranucleotide hypothesis" 1938 William Astbury obtains the first diffraction patters of DNA fibres
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
History of DNA
1944 Oswald Avery (Rockefeller Institute) proves that DNA carries thegenetic message by transforming bacteria
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
History of DNA
1950 Erwin Chargaff discovers A/G = T/C
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
History of DNA
1953 Watson and Crick propose the double helix as the structure of DNAbased on the work of Erwin Chargaff, Jerry Donohue, Rosy Franklinand John Kendrew
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
Maurice Wilkins – Kings College, LondonRichard Lavery
Institut de Biologie Physico-Chimique, Paris
•Watson-Crick model of DNA was based on X-ray •diffraction picture of DNA fibres
•(Rosalind Franklin and Maurice Wilkins)
•
•Rosalind Franklin
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
Rosalind Franklin (in Paris)Richard Lavery
Institut de Biologie Physico-Chimique, Paris
X-ray fibre diffraction pattern of B-DNARichard Lavery
Institut de Biologie Physico-Chimique, Paris
Linus Pauling’s DNARichard Lavery
Institut de Biologie Physico-Chimique, Paris
DNA secondary structure – double helix
•James Watson and Francis Crick, 1953- proposed a model for DNA structure
•DNA is the molecule of heredity (O.Avery, 1944)
•X-ray diffraction (R.Franklin and M. Wilkins)
•E. Chargaff (1940s) G = C and A = T in DNA
•
•
•Francis Crick Jim Watson
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
Watson and CrickRichard Lavery
Institut de Biologie Physico-Chimique, Paris
It has not escaped our notice …
It has not escaped our notice that the specific pairing we have postulated suggests a possible copying mechanism for the genetic material.
Richard Lavery
Institut de Biologie Physico-Chimique, Paris
Double helix ?Richard Lavery
Institut de Biologie Physico-Chimique, Paris
Nucleic Acids
DNA RNA
Central Dogma of Biology
DNA RNA Proteins Cellular Action
•transcription•translation•DNA
•re
plicati
on
•(deoxyribonucleic acids) •(ribonucleic acids)
•Natalia Tretyakova
•College of Pharmacy, U. of Minnesota
Dickerson Dodecamer (Oct. 1980)Richard Lavery
Institut de Biologie Physico-Chimique, Paris