Introduction to DNA Lecture notes edited by John Reif from PPT lectures by: Image from...

Introduction to DNAIntroduction to DNALecture notes edited by John Reif from PPT lectures by:Lecture notes edited by John Reif from PPT lectures by:

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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


DNA BASES

Nucleoside

Nucleotide

OH ribose

H deoxyribose

Richard Lavery


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


Base families

Purine (Pur / R) Pyrimidine (Pyr / Y)

C2

N1

C5C6N7

C4

C8

N9

N3

N1

C4

N3

C2

C5

C6

Richard Lavery


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



Pyrimidine BasesAll pyrimidine ring atoms lie in the same plane.

Richard B. Hallick


Pyrimidine Nucleotides



•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



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



Deoxyribose Sugar

The hydroxyl groups on the 5'- and 3'- carbons link to the phosphate groups to form the DNA backbone.

Richard B. Hallick


Richard B. Hallick


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:



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



DNA BASE PAIRING

Watson-Crick base pairs

Thymine -Adenine Cytosine -Guanine

Richard Lavery


Richard B. Hallick


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



Richard Lavery


Purine always binds with a Pyrimidine

Base pair dimensionsRichard Lavery


DNA/RNA chemical structure

DNA : A ,T,G,C + deoxyriboseRNA : A,U,G,C + ribose

Richard Lavery


DNA BACKBONE STRUCTURE

Richard B. Hallick


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

http://www.youtube.com/watch?v=ZGHkHMoyC5I

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


B-DNA Structure

Richard B. Hallick


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


B-DNA (lateral view)

R.H. helix

Richard Lavery


•Base stacking: an axial view of B-DNA



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.



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



•DNA can deviate from the ideal Watson-Crick structure

• Helical twist ranges from 28 to 42°

• Propeller twisting 10 to 20°

•Base pair roll



DNA grooves

MINOR

MAJOR

Richard Lavery


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



•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’



•B-type duplex is not possible for RNA

•steric “clash”

O

OH

HH

CH2

HO

HOBase

ribose

H



A-DNA STRUCTURE

A and B DNA allomorphs

B A

Hydration

Antiparallel strands

5’5’3’

3’

Richard Lavery


De-hydration

A-DNA (longitudinal view)Richard Lavery


A-DNA (lateral view)

R.H. helix

Richard Lavery


•A-form helix: dehydrated DNA; RNA-DNA hybrids

•Top View

•Right handed helix

• planes of bases are tilted

•20 ° relative the helix axis.


•

••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



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•



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

•• ••



Z-DNA STRUCTURE

Z-DNA (longitudinal view)Richard Lavery


Z-DNA (lateral view)

L.H. helix

Richard Lavery


Base pairs are rotated in Z-DNARichard Lavery


•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




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



Comparing A, B and Z-DNA

• 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



Backbone Dihedrals

Backbone dihedrals - I

0

Richard Lavery


+60°+10°

Dihedral angle definition

Staggered Eclipsed

Richard Lavery


Favoured conformations

gauche +

trans

gauche -

Richard Lavery


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


syn-anti glycosidic conformationsRichard Lavery


Sugar ring puckering

C5’

ENDO

EXO

Base

Richard Lavery


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


Sugar pucker and P-P distanceRichard Lavery


UNUSUAL DNA STRUCTURES

Alternative base pairs

Watson-Crick

Reversed Watson-Crick

Hoogsteen Reversed Hoogsteen

Richard Lavery


Watson-Crick + Hoogsteen = Base triplet

- note C(N3) protonation

Richard Lavery


Triple helix DNARichard Lavery


Guanine Hoogsteen pairing Base tetraplexRichard Lavery


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


Inverted repeat can lead to loop formationRichard Lavery


DNA cruciform

Holliday junction

Richard Lavery


PNA versus DNARichard Lavery


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


Parallel-stranded DNARichard Lavery


I-DNA: intercalated parallel-stranded duplexes Richard Lavery


and nucleotide anomers Richard Lavery


H OH is not the only change in passing from DNA to RNA .... Richard Lavery


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



•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


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


History of DNA

1944 Oswald Avery (Rockefeller Institute) proves that DNA carries thegenetic message by transforming bacteria

Richard Lavery


History of DNA

1950 Erwin Chargaff discovers A/G = T/C

Richard Lavery


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


Maurice Wilkins – Kings College, LondonRichard Lavery


•Watson-Crick model of DNA was based on X-ray •diffraction picture of DNA fibres

•(Rosalind Franklin and Maurice Wilkins)

•

•Rosalind Franklin



Rosalind Franklin (in Paris)Richard Lavery


X-ray fibre diffraction pattern of B-DNARichard Lavery


Linus Pauling’s DNARichard Lavery


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



Watson and CrickRichard Lavery


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


Double helix ?Richard Lavery


Nucleic Acids

DNA RNA

Central Dogma of Biology

DNA RNA Proteins Cellular Action

•transcription•translation•DNA

•re

plicati

on

•(deoxyribonucleic acids) •(ribonucleic acids)



Dickerson Dodecamer (Oct. 1980)Richard Lavery


Introduction to DNA Lecture notes edited by John Reif from PPT lectures by: Image from...

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