DNA: Structure, Dynamics and Recognition 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 Les Houches 2004

DNA: Structure, Dynamics and Recognition L1: Biological context, history, basic DNA structure Les Houches 2004

BIOLOGICAL CONTEXT

140 Mb 3300 Mb 4.4 Mb 0.6 Mb 4.6 Mb

50 mm Escherichia Coli, ≈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

500 Å E. coli membrane region © David S. Goodsell

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 mm (10-6 m) Bacterial cell 5 mm (10-5 m) Chromosome DNA 10 cm (10-1 m) Biological length scale

Biological length scale If 20 Å  1 cm then ... 1 m  5000 km ... Nucleus  15 m2 room Biological length scale

A "minimal" organism Hutchinson et al. Science 286, 1999, 2165 "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 Modelling the budding yeast cell cycle (Tyson & Novak)

580,000 bp 500 genes E-cell project

Molecular machines .... transcriptosome Nanobiotechnology D.S. Goodsell

Nucleosome

A LITTLE HISTORY ...

1865 Gregor Mendel publishes his work on plant breeding with the notion of "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 in the 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 the genetic 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 DNA based on the work of Erwin Chargaff, Jerry Donohue, Rosy Franklin and John Kendrew History of DNA

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-Crick base pairs

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. It has not escaped our notice …

Double helix ?

Dickerson Dodecamer (Oct. 1980)

DNA STRUCTURE

OH ribose H deoxyribose Nucleoside Nucleotide

Nucleotide triphosphates

Nucleotides are linked by phosphodiester bonds Strand has a direction (5'3')

DNA/RNA chemical structure RNA : A,U,G,C + ribose DNA : A ,T,G,C + deoxyribose DNA/RNA chemical structure

Base families Purine (Pur / R) Pyrimidine (Pyr / Y) C5 C4 N7 C6 C5 C6

Watson-Crick base pairs   Thymine -Adenine Cytosine -Guanine Watson-Crick base pairs

Base pair dimensions

CGCGTTGACAACTGCAGAATC

Hydration 3’ 5’ 5’ 3’ Antiparallel strands B A A and B DNA allomorphs

MAJOR MINOR DNA grooves

B-DNA (longitudinal view)

R.H. helix B-DNA (lateral view)

A-DNA (longitudinal view)

R.H. helix A-DNA (lateral view)

Z-DNA (longitudinal view)

L.H. helix Z-DNA (lateral view)

Base pairs are rotated in Z-DNA

n0 Backbone dihedrals - I

Dihedral angle definition +60° +10° Staggered Eclipsed Dihedral angle definition

Favoured conformations gauche + gauche - trans Favoured conformations

Backbone dihedrals - II : O3’ – P – O5’ – C5’ g- : P – O5’ – C5’ – C4’ t g : O5’ – C5’ – C4’ – C3’ g+ : C5’ – C4’ – C3’ – O3’ g+ e : C4’ – C3’ – O3’ – P t z : C3’ – O3’ – P – O5’ g- (Y) : O4’ – C1’ – N1 – C2 g- c(R) : O4’ – C1’ – N9 – C4 Backbone dihedrals - II

syn-anti glycosidic conformations

Baird & Tatlock 1901

Medicine Sets Manufactures by Messrs Burroughs, Wellcome & Co.

C5’ ENDO EXO Base Sugar ring puckering

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 Base tan P = (n4 - n1) - (n3 - n0) 2n2 (Sin 36° + Sin72°) Amp = n2 / Cos P Pseudorotation Equations Altona et al. J. Am. Chem. Soc. 94, 1972, 8205

Preferred sugar puckers

Sugar pucker and P-P distance

UNUSUAL DNA STRUCTURES

Alternative base pairs Reversed Watson-Crick Watson-Crick Hoogsteen Reversed Hoogsteen Alternative base pairs

Watson-Crick + Hoogsteen = Base triplet - note C(N3) protonation Watson-Crick + Hoogsteen = Base triplet

Triple helix DNA

Guanine Hoogsteen pairing  Base tetraplex

Quadruplex DNA

Inverted repeat can lead to loop formation

Holliday junction DNA cruciform

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 Peptide Nucleic acid(PNA)

Parallel-stranded DNA

I-DNA: intercalated parallel-stranded duplexes

a and b nucleotide anomers

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

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 Books on DNA