DNA Reactivity; Recombinant DNA I

1. DNA Reactivity; Recombinant DNA I Andy HowardIntroductory Biochemistry27 October 2014 DNA reactions; Recombinant I

2. DNA can be manipulated • We need to re-examine some of the reactivity of DNA • Much of our current understanding of molecular biology, and of the ways we can use it in medicine, agriculture, and basic biology, is derived from the kinds of genetic manipulations that we describe as recombinant DNA DNA reactions; Recombinant I

3. What we’ll discuss • DNA & RNA cleavage • Restriction Enzymes • Intercalation • Denaturation and renaturation of DNA • DNA density • DNA sequencing DNA reactions; Recombinant I

4. Why alkaline hydrolysis of RNA works • Cyclic phosphate intermediate stabilizes cleavage product DNA reactions; Recombinant I

5. The cyclic intermediate • Hydroxyl or water can attack five-membered P-containing ring on either side and leave the –OP on 2’ or on 3’. DNA reactions; Recombinant I

6. Consequences • So RNA is considerably less stable compared to DNA, owing to the formation of this cyclic phosphate intermediate • DNA can’t form this because it doesn’t have a 2’hydroxyl • In fact, deoxyribonucleic acid has no free hydroxyls on its sugar moieties! DNA reactions; Recombinant I

7. Enzymatic cleavage of oligo- and polynucleotides • Enzymes are phosphodiesterases • Could happen on either side of the P • 3’ cleavage is a-site; 5’ is b-site. • Endonucleases cleave somewhere on the interior of an oligo- or polynucleotide • Exonucleases cleave off the terminal nucleotide DNA reactions; Recombinant I

8. An a-specific exonuclease DNA reactions; Recombinant I

9. A b-specific exonuclease DNA reactions; Recombinant I

10. Specificity in nucleases • Some cleave only RNA, others only DNA, some both • Often a preference for a specific base or even a particular 4-8 nucleotide sequence (restriction endonucleases) • These can be used as lab tools, but they evolved for internal reasons DNA reactions; Recombinant I

11. Enzymatic RNA hydrolysis • Ribonucleases operate through a similar 5-membered ring intermediate:see bovine RNAse A: • His-119 donates proton to 3’–OP • His-12 accepts proton from 2’–OH • Cyclic intermediate forms with cleavage below the phosphate • Ring collapses, His-12 returns proton to 2’–OH, bases restored Bovine RNAseA13.6 kDa monomerPDB 1KF8, 1.15Å DNA reactions; Recombinant I

12. Variety of nucleases DNA reactions; Recombinant I

13. Restriction Endonucleases • Evolve in bacteria as antiviral tools • “Restriction” because they restrict the incorporation of foreign DNA into the bacterial chromosome • Recognize and bind to specific palindromic DNA sequences and cleave them • Self-cleavage avoided by methylation • Types I, II, III: II is most important • I and III have inherent methylase activity; II has methylase activity in an attendant enzyme DNA reactions; Recombinant I

14. What do we mean by palindromic? • In ordinary language, it means a phrase that reads the same forward and back: • Madam, I’m Adam. (Genesis 3:20) • Eve, man, am Eve. • Sex at noon taxes. • Able was I ere I saw Elba. (Napoleon) • A man, a plan, a canal: Panama! (T. Roosevelt) • With DNA it means the double-stranded sequence is identical on both strands DNA reactions; Recombinant I

15. Palindromic DNA • Example: G-A-A-T-T-C • Single strand isn’t symmetric: but the combination with the complementary strand is: • G-A-A-T-T-CC-T-T-A-A-G • These kinds of sequences are the recognition sites for restriction endonucleases. This particular hexanucleotide is the recognition sequence for EcoRI. DNA reactions; Recombinant I

16. Cleavages by restriction endonucleases • Breaks can be • cohesive (if off-center within the sequence) or • non-cohesive (blunt) (if they’re at the center) • EcoRI leaves staggered 5’–termini: cleaves between initial G and A • PstI cleaves CTGCAG between A and G, so it leaves staggered 3’–termini • BalI cleaves TGGCCA in the middle: blunt! DNA reactions; Recombinant I

17. iClicker question • 1. Which of the following is a potential restriction site? • (a) ACTTCA • (b) AGCGCT • (c) TGGCCT • (d) AACCGG • (e) none of the above. DNA reactions; Recombinant I

18. Example for E.coli • 5’-N-N-N-N-G-A-A-T-T-C-N-N-N-N-3’3’-N-N-N-N-C-T-T-A-A-G-N-N-N-N-5’ • Cleaves G-A on top, A-G on bottom: • 5’-N-N-N-N-GA-A-T-T-C-N-N-N-N-3’3’-N-N-N-N-C-T-T-A-AG-N-N-N-N-5’ • Protruding 5’ ends:5’-N-N-N-N-GA-A-T-T-C-N-N-N-N-3’3’-N-N-N-N-C-T-T-A-AG-N-N-N-N-5’ DNA reactions; Recombinant I

19. How often? • 4 types of bases • So a recognition site that is 4 bases long will occur once every 44 = 256 bases on either strand, on average • 6-base site: every 46= 4096 bases, which is roughly one gene’s worth DNA reactions; Recombinant I

20. EcoRI structure • Dimeric structure enables recognition of palindromic sequence •  sandwich in each monomer EcoRI pre-recognition complex PDB 1CL8 57 kDa dimer + DNA DNA reactions; Recombinant I

21. The biology problem • How does the bacterium mark its own DNA so that it does replicate its own DNA but not the foreign DNA? • Answer: by methylating specific bases in its DNA prior to replication • Unmethylated DNA from foreign source gets cleaved by restriction endonuclease • Only the methylated DNA survives to be replicated • Most methylations are of A & G,but sometimes C gets it too DNA reactions; Recombinant I

22. How this works • When an unmethylated specific sequence appears in the DNA, the enzyme cleaves it • When the corresponding methylated sequence appears, it doesn’t get cleaved and remains available for replication • The restriction endonucleases only bind to palindromic sequences • See Content Forum of Discussion Board for further details DNA reactions; Recombinant I

23. Methylases HhaI methyltransferasePDB 1SVU2.66Å; 72 kDa dimer • A typical bacterium protects its own DNA against cleavage by its restriction endonucleases by methylating a base in the restriction site • Methylating agent is generally S-adenosylmethionine DNA reactions; Recombinant I

24. Use of restriction enzymes • Nature made these to protect bacteria; we use them to cleave DNA in analyzable ways • Similar to proteolytic digestion of proteins • Having a variety of nucleases means we can get fragments in multiple ways • We can amplify our DNA first • Can also be used in synthesis of inserts that we can incorporate into plasmids that enable us to make appropriate DNA molecules in bacteria DNA reactions; Recombinant I

25. DNA is dynamic • Don’t think of these diagrams as static • The H-bonds stretch and the torsions allow some rotations, so the ropes can form roughly spherical shapes when not constrained by histones • Shape is sequence-dependent, which influences protein-DNA interactions DNA reactions; Recombinant I

26. Intercalating agents • Generally: aromatic compounds that can form -stack interactions with bases • Bases must be forced apart to fit them in • Results in an almost ladderlike structure for the sugar-phosphate backbone locally • Conclusion: it must be easy to do local unwinding to get those in! DNA reactions; Recombinant I

27. Instances of inter-calators DNA reactions; Recombinant I

28. Denaturing and Renaturing DNA See Figure 11.17 • When DNA is heated to 80+ degrees Celsius, its UV absorbance increases by 30-40% • This hyperchromic shift reflects the unwinding of the DNA double helix • Stacked base pairs in native DNA absorb less light • When T is lowered, the absorbance drops, reflecting the re-establishment of stacking DNA reactions; Recombinant I

29. Heat denaturation • Figure 11.14Heat denaturation of DNA from various sources, so-called melting curves. The midpoint of the melting curve is defined as the melting temperature, Tm.(From Marmur, J., 1959. Nature183:1427–1429.) DNA reactions; Recombinant I

30. GC content vs. melting temp • High salt and no chelators raises the melting temperature DNA reactions; Recombinant I

31. How else can we melt DNA? • High pH deprotonates the bases so the H-bonds disappear • Low pH hyper-protonates the bases so the H-bonds disappear • Alkalai is better: it doesn’t break the glycosidic linkages • Urea, formamide make better H-bonds than the DNA itself so they denature DNA DNA reactions; Recombinant I

32. What happens if we separate the strands? • We can renature the DNA into a double helix • Requires re-association of 2 strands: reannealing • The realignment can go wrong • Association is 2nd-order, zippering is first order and therefore faster DNA reactions; Recombinant I

33. Steps in denaturation and renaturation DNA reactions; Recombinant I

34. Rate depends on complexity • The more complex DNA is, the longer it takes for nucleation of renaturation to occur • “Complex” can mean “large”, but complexity is influenced by sequence randomness: poly(AT) is faster than a random sequence DNA reactions; Recombinant I

35. Second-order kinetics • Rate of association: -dc/dt = k2c2 • Boundary condition is fully denatured concentration c0 at time t=0: • c / c0 = (1+k2c0t)-1 • Half time is t1/2 = (k2c0)-1 • Routine depiction: plot c0t vs. fraction reassociated (c /c0) and find the halfway point. DNA reactions; Recombinant I

36. Wait. Can you do solve that differential equation properly? • Yes. Rewrite dc/dt = -k2c2 as –c -2dc = k2dt • Integrate both sides, with {-(-1)}c -1 = k2t+Q, i.e. 1/(k2t+Q) = cBoundary condition is c = c0 at t=0:-1/Q = c0, so c = 1/(k2t+1/c0),c/c0 = (k2c0t + 1)-1If t1/2 = time when c /c0 = ½,then t1/2 =(k2c0)-1, c /c0 = (t/t1/2+1)-1 DNA reactions; Recombinant I

37. Typical c0t curves DNA reactions; Recombinant I

38. Hybrid duplexes • We can associate DNA from 2 species • Closer relatives hybridize better • Can be probed one gene at a time • DNA-RNA hybrids can be used to fish out appropriate RNA molecules DNA reactions; Recombinant I

39. GC-rich DNA is denser • DNA is denser than RNA or protein, period, because it can coil up so compactly • Therefore density-gradient centrifugation separates DNA from other cellular macromolecules • GC-rich DNA is 3% denser than AT-rich • Can be used as a quick measure of GC content DNA reactions; Recombinant I

40. Density as function of GC content DNA reactions; Recombinant I

41. Tertiary Structure of DNA • In duplex DNA, ten bp per turn of helix • Circular DNA sometimes has more or less than 10 bp per turn - a supercoiled state • Enzymes called topoisomerases or gyrases can introduce or remove supercoils • Cruciforms occur in palindromic regions of DNA • Negative supercoiling may promote cruciforms DNA reactions; Recombinant I

42. DNA is wound • Standard is one winding per helical turn, i.e. 1 winding per 10 bp • Fewer coils or more coils can happen: • This introduces stresses that favors unwinding • Both underwound and overwound DNA compact the DNA so it sediments faster than relaxed DNA DNA reactions; Recombinant I

43. Linking, twists, and writhe • T=Twist=number of helical turns • W=Writhe=number of supercoils • L=T+W = Linking number is constant unless you break covalent bonds DNA reactions; Recombinant I

44. Examples with a tube DNA reactions; Recombinant I

45. How this works with real DNA DNA reactions; Recombinant I

46. How gyrases work • Enzyme cuts the DNA and lets the DNA pass through itself • Then the enzyme religates the DNA • Can introduce new supercoils or take away old ones DNA reactions; Recombinant I

47. Typical gyrase action • Takes W=0 circular DNA and supercoils it to W=-4 • This then relaxes a little by disrupting some base-pairs to make ssDNA bubbles DNA reactions; Recombinant I

48. Superhelix density • Compare L for real DNA to what it would be if it were relaxed (W=0): • That’sL = L - L0 • Sometimes we want = superhelix density= specific linking difference = L / L0 • Natural circular DNA always has  < 0 DNA reactions; Recombinant I

49.  < 0 and spools • The strain in  < 0 DNA can be alleviated by wrapping the DNA around protein spool • That’s part of what stabilizes nucleosomes DNA reactions; Recombinant I

50. Cruciform DNA • Cross-shaped structures arise from palindromic structures, including interrupted palindromes like this example • These are less stable than regular duplexes but they are common, and they do create recognition sites for DNA-binding proteins, including restriction enzymes DNA reactions; Recombinant I