DNA as a Nanostructure The central dogma:

1. DNA as a Nanostructure The central dogma:

2. The components of DNA and RNA:

3. DNA 5’-ACG -3’

4. DNA is double stranded -> double helix (2º structure)

5. The Minor and Major Groove of DNA Because the 2 glycosidic bonds are not diametrically opposite each other -> each base pair has a larger side -> the major groove -> and a smaller side -> the minor groove

6. Dimensions of DNA Adjacent bases are separated by 3.4 Å Helix repeats every 34 Å 10 bases per turn of helix Diameter of helix is 20 Å

7. Different forms of DNA Z- form Short oligonucleotides that have alternating pyrimidines and purines -> CGCGCGC

9. Other DNA structures than double helix - Quadruplex Just with Purin rich strands (G) Crystal structure of parallel quadruplexes from human telomeric DNA. The DNA strand (blue) circles the bases that stack together in the center around three co-ordinated metal ions (green). (By Thomas Splettstoesser)

10. Other DNA structures than double helix - Quadruplex The arrangement of guanine bases in the G-quartet, shown together with a centrally placed metal ion. Hydrogen bonds are shown as dotted lines, and the positions of the grooves are indicated. (b) The poly(dG) four-fold, right-handed helix. (c) Surface view representation of a quadruplex structure comprising eight G-quartets, with the central channel exposed to show an array of metal ions (coloured yellow). -> Microelectronics

11. Relaxed and Supercoiled DNA (3º structure) • Supercoiled DNA -> Relaxed DNA • -> 1 DNA strand needs to be nicked • Enzymes (Topoisomerases) • Sheer forces or chemicals

12. DNA Supercoiling • -> The twist is the number of helical turns in the DNA • -> the writhe is the number of times the double helix crosses over on itself (these are the supercoils). • The relationship of twist, writhe and supercoiling (Linking number)is expressed as the equation: • S = T + W • Extra helical twists are positive and lead to positive supercoiling, while subtractive twisting causes negative supercoiling. -> Overtwisting leads to   postive supercoiling -> undertwisting leads to negative supercoiling

13. Linking Number Determines the Degree of Supercoiling Linking Number describes the linking of two closed curves in three-dimensional space

14. Linking Number Determines the Degree of Supercoiling Extra helical twists are positive and lead to positive supercoiling, while subtractive twisting causes negative supercoiling. -> Overtwisting leads to   postive supercoiling -> undertwisting leads to negative supercoiling Most DNA molecules are neg. supercoiled

15. Supercoiling in eukaryotic linear DNA (Chromosomes) -> Supercoiling happens between Histone proteins

16. Nucleic Acid Synthesis - Polymerization DNA polymerase -> Replication: DNA -> DNA 5’ -> 3’ Primer Proof reading RNA polymerase -> Transcription: DNA -> RNA 5’ -> 3’ No primer No proof reading

17. DNA Replication, Recombination, and Repair

18. DNA replication Replication is: -> bidirectional -> semiconservative

19. DNA Replication

20. Topoisomerase I structure/mechanism • Topoisomerases prepare double helix for unwinding -> change “linking number” • Preparation for unwinding requires neg. supercoiled DNA (TopoII) • In the process of unwinding -> DNA needs to be relaxed (TopoI) • -> Important for Replication, Transcription, Recombinantion • TopoI -> cleaves one strand -> relaxes neg. supercoiled DNA No Energy (ATP) required !!

21. Topoisomerase II structure/mechanism (DNA gyrase) TopoII -> cleaves both strands -> introduces negative supercoiling -> Requires Energy (ATP) !!! Gyrase Inhibitors

22. E. coli DNA polymerase Polymerase Activity α-subunit ε-subunit

23. DNA polymerase mechanism One Mg2+ coordinates the 3’-OH group of the primer -> OH-group of primer attacks P-group of nucleotide Polymerase donates 2 H-bond to base pair in minor groove

24. Shape selectivity Binding of NTP induces conformational change -> generating a tight pocket for base pair -> Conformational change just when incoming dNTP fits to template DNA

25. Proofreading ε Subunit of E. coli Polymerase III (responsible for sythesis of DNA) has 3’-> 5’ exonulease activity The growing chain moves sometimes to the exonuclease site -> checking if incorrect nucleotide is incorporated -> wrong nucloetide is hydrolysed

26. Helicase structure/mechanism Used for unwinding of DNA -> replication It has 4 domains: A1 domain has a P-loop NTPase -> bind ATP Release of ADP -> cleft between B1 + A1 opens -> A1 still bond tighter to DNA -> DNA pulled across B1 towards A1 Binding of ATP -> conformational change of P-loop -> closure of cleft between B1 + A1 -> A1 releases DNA -> A1 slides along DNA -> moving closer to B1 B1 + A1 bind ss DNA

27. Origin of Replication (E. coli) Surrounding of Ori -> AT rich -> important for local melting of DNA Binding and assembly of DnaA initiates replication

29. Prokaryotes Eukaryotes Initiation of replication: Origins In Prokaryotes: 1 folk/circular chromosom -> 1000 bp/sec -> 42 min to replicate E. coli chromosome (4.6 mill bp) In Eukaryotes: In humans -> 100 bp/sec per folk Size: 3x109 bp in 23 chromosomes Assume the same as in prokaryotes: 1 folk per chromosome -> 23 folks -> 1.3x108 bp/folk with a speed of 100 bp/sec 1 Replication cycle would take 1.3x106 sec -> 362 h-> 15 days !!!! -> in real: 1 replication cycle is 8 h -> 30.000 folks (not all always active)

30. Elongation of Replication Introduces neg. supercoiling DNA polymerase I degrades RNA primer DNA ligase closes the fragments

31. DNA polymerase complex -> Replisome DNA enclosing site DNA polymerase III core enzyme -> dimer

32. Coordination Between the Leading and the Lacking Strand -> Replication is bidirectional

35. Telomeres Eukaryotes need Telomer ends -> otherwise chromosomes would shrink each replication cycle In the process -> quadruplex structures are formed Telomer end are involved in aging of cell !!

37. Recombination Essential in the following processes: -> When replication stops -> recombination reset replication process -> When DNA strands break -> recombination repairs DNA -> In meiosis -> genetic diversity (cross over events) -> generation of diversity for antibodies (Exon shuffling) -> viruses use recombination to integrate into genome -> in recombinant technologies -> generate recombinant organisms (knock-out mice)

38. RecA protein initiates recombination by strand invasion

39. Recombination mechanism – Holliday Junction

40. Mutations – changes in DNA sequence Transition: AT <-> GC TA <-> CG Exchange of Purine by Purine and Pyrimidine by Pyrimidine Transversion: AT <-> TA AT <-> CG TA <-> GC CG <-> GC Exchange of Purine by Pyrimidine and the other way round

42. Mutation by Tautomerization Tautomerization: The conversion of two isomers that differ only in the position of protons (and often double bonds) -> Transition from AT -> GC -> Transition from TA -> CG Analog to thymine

45. Chemical Mutagens Transition from AT -> GC Conversion of A -> Hypoxanthine Hypoxanthine pairs with C Acridines: Induce frame shift by intercalating into DNA leading to incorporation of additional bases (Ethidium bromide)

49. Chemical Mutagens Produced by fungi; Activated by Cyt P450 Modifies bases such as G -> Transversion from GC -> TA Active epoxide

50. Deamination of modified Cytosine 5-methylcytosine -> hot spot for mutations