1. Structures of nucleic acids II Southern blot-hybridizations Sequencing Supercoiling: Twisting, Writhing and Linking number

2. Southern blot-hybridizations Allows the detection of a particular DNA sequence among the many displayed on an electrophoretic gel. e.g. determine which among many restriction fragments contains a gene. Transfer the size-separated DNA fragments out of the agarose gel and onto a membrane (nylon or nitrocellulose) to make an immobilized replica of the gel pattern. Hybridize the membrane to a specific, labeled nucleic acid probe and determine which DNA fragments contain that labeled sequence.

3. Steps in Southern blot-hybridization

4. Steps in Southern blot-hybridization, continued

5. Sizes of chromosomes

6. Sizes of chromosomes, cont’d

7. Strategy to determine DNA or RNA sequence Generate a nested set of fragments with one common, labeled end The other end terminates at one of the 4 nucleotides Electrophoretic resolution of the fragments allows the reading of the sequence: Fragment of length 47 ends at G 48 A 49 T Sequence is …GAT….

8. Common sequencing techniques DNA: Maxam & Gilbert DNA: Sanger RNA Chain termination by dideoxy- nucleotides Base-specific chemical cleavage Nucleotide-specific enzymatic cleavage Restriction endonuclease Primer for DNA polymerase Natural end of RNA 32 P 32 P or fluores- cence 32 P TechniqueCommon endLabelNt-specific end

9. Example of dideoxynucleotide sequencing Reactions: Fig. 2.30 Output: Fig. 2.31 Cycle Sequencing Movie: –http://vector.cshl.org/resources/BiologyAnimationLibrary.htm The Sanger dideoxynucleotide method is amenable to automation performed by robots. This approach is the one adapted for virtually all the whole-genome sequencing projects.

10. Example of output from automated dideoxysequencing

11. Supercoiling of topologically constrained DNA Topologically closed DNA can be circular (covalently closed circles) or loops that are constrained at the base The coiling (or wrapping) of duplex DNA around its own axis is called supercoiling.

12. Different topological forms of DNA Genes VI : Figure 5-9

13. Negative and positive supercoils Negative supercoils twist the DNA about its axis in the opposite direction from the clockwise turns of the right-handed (R-H) double helix. –U nderwound (favors unwinding of duplex). –Has right-handed supercoil turns. Positive supercoils twist the DNA in the same direction as the turns of the R-H double helix. –Overwound (helix is wound more tightly). –Has left-handed supercoil turns.

14. Components of DNA Topology : Twist The clockwise turns of R-H double helix generate a positive Twist (T). The counterclockwise turns of L-H helix (Z form) generate a negative T. T = Twisting Number B form DNA: + (# bp/10 bp per twist) A form NA: + (# bp/11 bp per twist) Z DNA: - (# bp/12 bp per twist)

15. Components of DNA Topology : Writhe W = Writhing Number Refers to the turning of the axis of the DNA duplex in space Number of times the duplex DNA crosses over itself Relaxed molecule W=0 Negative supercoils, W is negative Positive supercoils, W is positive

16. Components of DNA Topology : Linking number L = Linking Number = total number of times one strand of the double helix (of a closed molecule) encircles (or links) the other. L = W + T

17. L cannot change unless one or both strands are broken and reformed A change in the linking number,  L, is partitioned between T and W, i.e.  L=  W+  T if  L = 0, then  W= -  T

18. Relationship between supercoiling and twisting Figure from M. Gellert; Kornberg and Baker

19. DNA in most cells is negatively supercoiled The superhelical density is simply the number of superhelical (S.H.) turns per turn (or twist) of double helix. Superhelical density =  = W/T = -0.05 for natural bacterial DNA –i.e., in bacterial DNA, there is 1 negative S.H. turn per 200 bp (calculated from 1 negative S.H. turn per 20 twists = 1 negative S.H. turn per 200 bp)

20. Negatively supercoiled DNA favors unwinding Negative supercoiled DNA has energy stored that favors unwinding, or a transition from B-form to Z DNA. For  = -0.05,  G=-9 Kcal/mole favoring unwinding Thus negative supercoiling could favor initiation of transcription and initiation of replication.

21. Topoisomerase I Topoisomerases: catalyze a change in the Linking Number of DNA Topo I = nicking-closing enzyme, can relax positive or negative supercoiled DNA Makes a transient break in 1 strand E. coli Topo I specifically relaxes negatively supercoiled DNA. Calf thymus Topo I works on both negatively and positively supercoiled DNA.

22. Topoisomerase I: nicking & closing Genes VI : Figure 17-15 One strand passes through a nick in the other strand.

23. Topoisomerase II Topo II = gyrase Uses the energy of ATP hydrolysis to introduce negative supercoils Its mechanism of action is to make a transient double strand break, pass a duplex DNA through the break, and then re- seal the break.

24. TopoII: double strand break and passage

25. Quiz:  W during B to Z transition A negatively supercoiled DNA molecule undergoes a B to Z transition over a segment of 360 base pairs. What is the effect on the writhing (supercoiling)?

26. Quiz:  W during B to Z transition A negatively supercoiled DNA molecule undergoes a B to Z transition over a segment of 360 base pairs. What is the effect on the writhing (supercoiling)? The molecule is not opened during this transition, so the linking number does not change.  L = 0 The twist changes from that in B-form (T B ) to that in Z DNA (T Z ):  T = T Z - T B = - 30 - (+36) = -66  W = -  T = -(-66) = +66 T B = 360/ +10 = +36T Z = 360/-12 = -30