Two types of interactions stabilize the DNA double helix: base pairing and base stacking

1. Fluctuational Opening of Base Pairs in DNAMaxim Frank-KamenetskiiBoston Universitymfk@bu.edureprints at: www.bu.edu/cab

2. Two types of interactions stabilize the DNA double helix: base pairing and base stacking Being a nanoscale object, DNA must experience thermal fluctuations, or breathing: Example: 3 base pairings and 4 base-stackings are disrupted

3. Why is it important? • Due to the breathing, reactive groups normally buried inside the double helix become accessible for chemicals and proteins • Nuclear magnetic resonance (NMR) makes it possible to study base-pair breathing for very short DNA helices thus providing with direct experimental data to compare with a prediction algorithm • Base-pair opening is very important for DNA damage, mutations and repair

4. Base-pair opening is very important for DNA damage, mutations and repair Example: Uracil DNA Glycosylase flips out uracil from the double helix Parikh et al. PNAS, 2000

5.  imino protons Watson - Crick Base Pairs 

6. DNA stability with respect to melting To separate base-pairing and base-stacking contribution into the melting free energy, one needs to determine independently either stacking or base-pairing • Base pairing: DGBPA and DGBPG • Stacking: DGSTKL 1 1 ST BP BP D = D + D + D G G G G KL KL K L 2 2

7. Nicked DNA X-ray crystallography, NMR, EM, PAGE, thermal denaturation studies agree: Nick introduces only minor perturbations to the structure of the DNA double helix. Really?

8. DNA fragments with solitary nicks or gaps Nicks are introduced enzymatically by nicking endonucleases N.BstNBI N.AlwI Nicks are located in the KL/K’L’ dinucleotide stack in the DNA fragment N.BstNBI N.BbvCIA Gaps (2-nt or longer) are obtained by consecutive digestion by two nicking enzymes

9. Relative mobility 1.00 0.95 Urea concentration, M 0.90 0.85 0.80 1 2 3 4 5 6 7 pGC pCC pCA pTA pG2 Gel electrophoresis of nicked DNA • DNA fragments carrying a single nick • are somewhat retarded with respect to intact fragments • retardation is enhanced by addition of denaturant (urea) to the gel • degree of retardation depends • on the identity of nicked dinucleotide stack 5 ' GC GG AC TC FOR CG AG 2 bp 10 bp M 3 ' CG CC TG AG GC TC REV gap gap 2.3 M 3.5 M 4.9 M 7.0 M

10. 0.90 0.85 0.80 0.75 0.70 1.2 M relative mobility 1 2 3 4 5 6 7 gap size, nt Stacked-Unstacked Equilibrium in nicked DNA DGST mclosed mopen Unstacked or open conformation: kinked DNA Stacked or closed conformation: straight DNA molecule; moves fast We assume that the kinked molecule moves like gapped DNA: æ ö ST N G m - m D closed ç ÷ open = - exp m = ÷ ç - m N RT è ø open closed m is the gel-electrophoretic mobility

11. GKLG CKLC '' GKLG CKLC '' Standard free energies are estimated by extrapolating DGST(urea) to zero AG AA CG TG TC GC AC GG 1 0 -1 Free energy calculated from mobility data -2 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 AT GA CC CA TA TT 1 GT CT 0 -1 -2 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 Urea concentration, M

12. -GKLG- -CKLC- -GKLG- -CKLC- '' '' We obtain 32 stacking parameters 0 l o m -1 / l a c k -2 , L T K S G -3 D GC GG CC CG AC GT TC GA AG CT TG CA AT AA TT TA DGST values describing the equilibrium at the site of the DNA nick are equivalent to the stacking parameters of intact double helix

14. 400 K , e 390 r u t 380 a r e p 370 m e 360 t g 350 n i t l e 340 m 0.0 0.2 0.4 0.6 0.8 1.0 GC content Determination of base-pairing contribution to melting free energy • Base pairing: DGBPA and DGBPG • Stacking: DGSTKL 1 1 ST BP BP D = D + D + D G G G G KL KL K L 2 2 Marmur-Doty plot

15. DNA differential melting curves

16. l o -0.5 m / l -1.0 a c k -1.5 , t n -2.0 a i r a -2.5 v I I I I I I I n i 3 5 6 8 1 2 4 7 Free energy of DNA melting DGKL • Base pairing: DGBPA and DGBPG • Stacking: DGSTKL 1 1 ST BP BP D = D + D + D G G G G KL KL K L 2 2 There are 16 KL contacts, 10 of them are different; only their 8 invariants can be determined from melting experiments with long DNA molecules: invariants I1 = DGAA I2 = DGGG I3 = DGAT + DGTA I4 = DGGC + DGCG I5 = DGAC + DGCA I6 = DGAG +DGGA I7 = DGAT + DGTC+ DGCA I8 = DGAG + DGGC+ DGCA I

17. AA AA TA TA TT TT AT AT 0.0 CG GC -0.5 22 °C GG CC -1.0 G -1.5 GC CG -2.0 N -2.5 30 35 40 45 50 55 I temperature, °C 0.5 TA AT 0.0 32 °C AA G TT -0.5 AT TA -1.0 N -1.5 -2.0 10 20 30 40 50 I temperature, °C 42 °C G N I G 52 °C N I Temperature Dependence individual stacks

18. Temperature dependenceof stacking andbase-pairing

19. Salt dependenceof stacking andbase-pairing

20. Theoretical prediction of bp opening probability Partition function for DNA: Z = ak dk dk+1 x = Kd Z Base-pairing Stacking x Ring factor (adjustable parameter)

21. NMR data from Gueron & Leroy and Russu groups DNA Breathing NMR Theory

22. 4 30 15 b 15 °C 30 °C 5 °C 37 °C p o p e 1.0 3 n i n 20 10 g p r 2 o b a 0.5 b 10 5 i l i 1 t y x 1 0 5 0 C3 A4 G5 A6 C3 A4 G5 A6 C3 A4 G5 A6 C3 A4 G5 A6 Temperature dependence of bp opening NMR (Russu group) Theory x = 2.3·10-3 for all temperatures

23. Conclusions • Stacking rather than base-pairing determines the stability of the double helix with respect to melting • DNA breathing probability can be predicted by an algorithm based on the partition function calculation • Since stacking dominates, the opening probability strongly depends on the nearest neighbors

24. Acknowledgements • Katya Protozanova • Peter Yakovchuk • Andy Krueger