DNA: Structure, Dynamics and Recognition Les Houches 2004 Richard Lavery Institut de Biologie...

DNA: Structure, Dynamics DNA: Structure, Dynamics and Recognitionand Recognition

Les Houches 2004

Richard Lavery

Institut de Biologie Physico-Chimique, Paris

Les Houches 2004

L1: Biological context, history, basic DNA structure

L2: Introductory DNA biophysics and biology

L3: DNA dynamics

L4: DNA deformation

L5: Recognizing DNA

DNA: Structure, Dynamics DNA: Structure, Dynamics and Recognitionand Recognition

Les Houches 2004

L1: Biological context, history, basic DNA structure

BIOLOGICAL CONTEXT

140 Mb

3300 Mb

0.6 Mb

4.4 Mb

4.6 Mb

VEVRLREDPETFLVQLYQHCPPLARIDSVEREPFIWSQLPTEFTIRQSTGGTMNTQIVP FT

DAATCPACLAEMNTPGERRYRYPFINCTHCGPRFTIIRAMPYDRPFTVMAAFPLCPACD FT

KEYRDPLDRRFHAQPVACPECGPHLEWVSHGEHAEQEAALQAAIAQLKMGKIVAIKGIG FT

GFHLACDARNSNAVATLRARKHRPAKPLAVMLPVADGLPDAARQLLTTPAAPIVLVDKK FT

YVPELCDDIAPDLNEVGVMLPANPLQHLLLQELQCPLVMTSGNLSGKPPAISNEQALAD FT

LQGIADGFLIHNRDIVQRMDDSVVRESGEMLRRSRGYVPDALALPPGFKNVPPVLCLGA FT

DLKNTFCLVRGEQAVLSQHLGDLSDDGIQMQWREALRLMQNIYDFTPQYVVHDAHPGYV FT

SSQWAREMNLPTQTVLHHHAHAAACLAEHQWPLDGGDVIALTLDGIGMGENGALWGGEC FT

LRVNYRECEHLGGLPAVALPGGDLAAKQPWRNLLAQCLRFVPEWQNYSETASVQQQNWS FT

VLARAIERGINAPLASSCGRFFDAVAAALGCAPATLSYEGEAACALEALAASCHGVTHP FT

VTMPRVDNQLDLATFWQQWLNWQAPVNQRAWAFHDALAQGFAALMREQATMRGITTLVF

Escherichia Coli, ≈4.6 Mb

50 m

E. coli membrane region© David S. Goodsell

500 Å

• 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

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)

Biological length scale

If 20 Å 1 cm then ...

1 m 5000 km ...

Nucleus 15 m2 room

A "minimal" organism

"We are wondering if we can come up with a molecular definition of life"

"The goal is to fundamentally understand the components of the most basic living cell"

Craig Venter, founder of Celera Genomics, IBEA and several other gene tech companies

Hutchinson et al. Science 286, 1999, 2165

Modelling the budding yeast cell cycle (Tyson & Novak)

K.C. Chen et al. Mol. Biol. Cell Cycle 11 (2000) 369

E-cell project

580,000 bp

500 genes

Molecular machines .... transcriptosome

Nanobiotechnology D.S. Goodsell

Nucleosome

A LITTLE HISTORY ...

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 

History of DNA

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

History of DNA

1950 Erwin Chargaff discovers A/G = T/C 

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

Maurice Wilkins – Kings College, London

Rosalind Franklin (in Paris)

X-ray fibre diffraction pattern of B-DNA

Linus Pauling’s DNA

Watson-Crick base pairs

Thymine -Adenine Cytosine -Guanine

Watson and Crick

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.

Double helix ?

Dickerson Dodecamer (Oct. 1980)

DNA STRUCTURE

Nucleoside

Nucleotide

OH ribose

H deoxyribose

.

O

H

H

OH

H

H

H

O N

N

NH2

CH2P O

O

O -

P O

O

O -

P O- O

O

O - 1'

2'3'

4'

5'

2'-deoxycytidine 5' triphosphate

O

H

H

H

H

CH2P O

O

O -

P O

O

O -

P O- O

O

O - 1'

2'3'

4'

5'

N

N

NH2

N

N

2'-deoxyadenosine 5' triphosphate

OH H

O

H

H

OH

H

H

H

CH2P O

O

O -

P O

O

O -

P O- O

O

O - 1'

2'3'

4'

5'

N

NHN

N

O

H2N

2'-deoxyguanosine 5' triphosphate

O

H

H

OH

H

H

H

CH2P O

O

O -

P O

O

O -

P O- O

O

O - 1'

2'3'

4'

5'

O N

H N

O

CH3

thymidine 5' triphosphate

Nucleotide triphosphates

Nucleotides are linked by phosphodiester bonds

Strand has a direction (5'3')

DNA/RNA chemical structure

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

Base families

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

C2

N1

C5C6N7

C4

C8

N9

N3

N1

C4

N3

C2

C5

C6

Watson-Crick base pairs

Thymine -Adenine Cytosine -Guanine

Base pair dimensions

34 Å

3.4 Å

20 Å

MinorGroove

MajorGroove

GC

CG

AT

TA

CG

GCAT

TA

TA

AT

GCCG

GC

Strands areantiparallel

CGCGTTGACAACTGCAGAATC

A and B DNA allomorphs

B A

Hydration

Antiparallel strands

5’5’3’

3’

DNA grooves

MINOR

MAJOR

B-DNA (longitudinal view)

B-DNA (lateral view)

R.H. helix

A-DNA (longitudinal view)

A-DNA (lateral view)

R.H. helix

Z-DNA (longitudinal view)

Z-DNA (lateral view)

L.H. helix

Base pairs are rotated in Z-DNA

Backbone dihedrals - I

0

+60°+10°

Dihedral angle definition

Staggered Eclipsed

Favoured conformations

gauche +

trans

gauche -

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

syn-anti glycosidic conformations

Baird & Tatlock 1901

Medicine SetsManufactures by Messrs Burroughs, Wellcome & Co.

Sugar ring puckering

C5’

ENDO

EXO

Base

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 puckers

Sugar pucker and P-P distance

UNUSUAL DNA STRUCTURES

Alternative base pairs

Watson-Crick

Reversed Watson-Crick

Hoogsteen Reversed Hoogsteen

Watson-Crick + Hoogsteen = Base triplet

- note C(N3) protonation

Triple helix DNA

Guanine Hoogsteen pairing Base tetraplex

Quadruplex DNA

Inverted repeat can lead to loop formation

DNA cruciform

Holliday junction

PNA versus DNA

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

Parallel-stranded DNA

I-DNA: intercalated parallel-stranded duplexes

and nucleotide anomers

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

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