Key Points. • NMR dynamics divided into 2 regimes: fast and slow. • How protein
mojons affect NMR parameters depend on whether they are faster or slower.
A vs. B vs. Z DNA, Triplexes and Quadruplexes
Dickerson Dodecamer (bdl001.pdb)
Arnott A RNA Structure (arn0035.pdb)
Comparison of groove width and depth A DNA B DNA
MAJOR MAJOR minor
minor
MAJOR MAJOR minor
Relationship between sugar pucker and helix type B DNA
P O
A DNA
P Base
O
O
Base O
O
7.0 A
P
O
P
5.9 A C3'- endo, 3E
C2'- endo, 2E Orientation of base pairs relative to helix axis 3.4 A
h = 3.4 A
3.4 A
h=3A
Contrasting A and B Helices helical repeat displacement, d helical rise/residue, h tilt twist propellar twist sugar pucker intra chain P-P distance Major groove width depth minor groove width depth
A helix 11 4.4 2.6-3.3 10-20 30-33
B helix 10.5 -0.2 - -1.8 3.4 -6 36-45
C3’-endo, 3E 5.9
C2’-endo and others, 2E 7.0
2.7 13.5
11.7 8.5
11 2.8
5.7 7.5
h
helical repeat
Table: AvsB.doc
Consequences of propeller twisting on local B DNA structure and how DNA responds
minor groove clash at 5'-Py-Pu-3'
major groove clash at 5'-Pu-Py-3'
minimizing minor groove clash at 5'-Py-Pu-3'
Shifting BP over to minimize clashes
Clash due to large twist angle
Clash minimized by decreasing twist angle
Sequence-Dependent Variation of DNA Structure: Callidine’s rules Local DNA structure can be viewed as a result of two opposing effects: 1. The drive to optimize H-bonding and base stacking interactions. 2. The drive to minimize unfavorable VDW interactions between purines that result from the positive propellar twisting of the base pairs at Pu-Py (RY) and Py-Pu (YR) sequences.
DNA compensates for the clashes by both local and coupled responses: 1. Local Responses a. Decrease propellar twist to 0 b. Slide base pair over (reduce δ) 2. Coupled Responses a. Reduce relative twist b. Reduce relative roll angle
Relative magnitude of responses Major Groove
Minor Groove
local responses (at a base pair)
R Y
Y R
propellar twist (always decrease)
-1 -1
-2 -2
δ (open purine δ, decrease pyrimidine δ)
+1 -1
-2 +2
coupled responses (between base pairs)
xRYx
x Y R x
twist angle (always reduce)
+1-2 +1
+2 -4 +2
+1 -2 +1 roll angle (+ value defined as opening in the major groove; changing the central roll angle must be equally compensated by the adjacent roll angles)
-2 +4 -2
εL-εR
+3
-5
240 260 280 300 wavelength (nm)
CD of poly (dG-dC) at pH 7 25 oC Solid line: 0.2 M NaCl Dotted line: after addition of more NaCl
Rich hexamer Z DNA structure (zdf002.pdb)
Rich hexamer Z DNA structure (zdf002.pdb) Minor Groove
Major Groove
Rich hexamer Z DNA structure (zdf002.pdb) CpG step GpC step C3G4-C9G10 G4C5-G8C9
Interstrand base stacking
Intrastrand base stacking
syn anti
Structural Features of Z DNA 1. No major groove, just a surface 13.8 Α wide, 2 A deep. 2. Very deep and narrow minor groove, 3.7 A wide, 8.8 A deep. 3. 18 A wide helix (19 A for B, 23 for A) 4. Interchain phosphate-phosphate distance of 7.7 A; high charge density. 5. 12 bp/turn, 7.4 A/dinucleotide repeat 6. Watson Crick base pairing between C and G. 7. Dinucleotide repeat with an intrastrand GC pi stack at 5’GC-3’ steps and an interstand CC pi stack at 5’-CG-3’ steps. 8. Left-handed stacking at 5’-GC-3’ site with at twist of 50o and -15o twist at 5’-CG-3’ steps. 9. Helix axis is dislocated at 5’-CG-3’ steps. 10. G is in a syn glycosyl conformation, C is in an anti conformation.
Flipping of Base Pairs in B to Z transition TOP VIEW 5'-down O
O
N O
N
5'-up O
N
N H N
anti
O
H H N
O
N H
anti
H
3'-up
O
N
O
Base pair flips by 180o
3'-up
3'-down 3'-down
5'
O
O
O H O
N H
O
N H
N
N
N
N N
O
H N
O O
5'-up H
SIDE VIEW 5' O
O
O
G
glycosyl bond rotates
C
O
O
5'
O O
3' whole nucleoside rotates
O
O
G
C
O O O
3'
Conformational Transition Between B and Z DNA
a a a a a a a a
3'
5' a a a a a a a a
GC CG GC CG GC CG GC CG
5'
B DNA =
3' O
s a s a s a s a
3'
5' GC CG GC CG GC CG GC CG
5'
Z DNA
a= anti glycosyl s = syn glycosyl
a s a s a s a s
3'
A nanomechanical device based on B to Z transtion
Donor and acceptor molecules (fluorescein and Cy3) are attached to a DNA molecule containing a (GC)20 section. When in B form the two dyes are close and show strong FRET, when in Z form, the DNA unwinds by about 3.5 turns, and extends about 6 A, changing the distance by 20-60 A, and greatly lowers the FRET. Nature. 1999, 144-6
Minimum Salt Concentration Required to Form Z DNA Ion (mM)
poly d(G-C)
poly d(G-m5C)
Na+
2500
700
Mg2+
700
0.6
Ca2+
100
0.6
Ba2+
40
0.6
Co(NH3)63+
0.02
0.005
EtOH
60% v/v
20%
Mg2+ + 10% EtOH 4 mM
-
Mg2+ + 20% EtOH 0.4 mM
-
O O P O H
O
H O
O
O
P O
O-
H H N
OH2 O
Mg O H H H2O OH2
H2O
C
O
N N
N
N
H
O
O
Structural factors favoring Z DNA formation CH3 H dR
H
O
N N
N H
N
N H
m 7G
m 5C
electrostatics (Z DNA has higher charge density)
hydrophobicity (methyl group occupies hydrophobic pocket)
O O
N N
N N
O
H
O
RO
dR
O
H
N
H
N
N
CH3
CH3
H N
H N
N
H N
H
RO
bad steric interactions in the anti conformation
N
H
H
N
RO
O
N
N CH3
O
RO
less severe steric interactions in the syn conformation which is the conformation at the purine site in Z DNA
Effect of Substituents on DNA Conformation H
H H dR
O
N N
N H
H N
H
H
N
H
N
N N H
O
dR
N N
N H
O
N H
N
N H
H
2-aminopurine H H dR
O
N N
N H
H N
N
inosine
H
N
H N
O
H N
N H
dR
H
dR
Polymer
C2-NH2 group
Helix
d(A)•d(T)
no
B
d(I)•d(C)
no
B
d(IIT)•d(ACC)
no
B
d(AG)•d(CT)
yes
B, A
d(AGC)•d(GCT)
yes
B, A
d(GC)
yes
B, A, Z
d(GT)
yes
B, A, Z
d(2AP-T)
yes
B, A, Z
O
dR
Metal cations bind in the minor groove with water Structure of the potassium form of CGCGAATTCGCG: DNA deformation by electrostatic collapse around inorganic cations. Biochemistry. 1998 Dec 1;37(48):16877-87.
Figure 1 The fused hexagon motif of A-tract DNA. The four layers are coded by color with the primary layer light blue, the secondary layer magenta, the tertiary layer blue, and the quaternary layer red. The fused hexagon motif is shown in space filling representation, with van der Waal radii of oxygen atoms. (a) Stereoview into the minor groove of the DNA. The DNA is colored by CPK and shown in stick representation. (b) View across the groove, approximately down the normal of the central hexagons. Sites of potassium occupancy are indicated by plus signs. The DNA bases are shaded. Base functional groups that interact with the fused hexagon motif are indicated by circles. (c) The geometry of the sodium form fused hexagon motif. Distances are in red and angles are in white.
X-ray crystal structure of a 1:1 complex of netropsin: DNA
Loss of O2 carbonyl disrupts spine of hydration
The role of minor groove functional groups in DNA hydration. Nucleic Acids Res. 2003 Mar 1;31(5):1536-40.
Conformational and Electrostatic Factors Favoring Various Forms of DNA or RNA B Form of RNA
P O
A Form of RNA
P O
Base O
Base
bad steric interaction
O
O
P
P OH
C3'- endo, 3E
C2'- endo, 2E
O P
H
O
Z DNA intrastrand phosphate hydration
H
O
O
Z: 5.6 A
P
H
O H
O
O
A: 5.7 A B: 6.7 A
steric interaction less severe
O
OH
Z: 6.2 A
P
O
Evidence for Triplex Helix Formation From Mixing Experiments Monitored by UV
nd(T)10d(A)10
d(T)10 + d(A)10 A260 1
A260 1
0.9
0.9
0.8
0.8
0.7
0.7
0.6
0.6
0
50 molar % dT10
100
0
50 66
100
molar % dT10
Analysis of mixing curves of nucleic acids by UV relies on the hypochromic effect observed upon formation of stacked base pairs
Polypyrimidine Triplex Motif H CH3
N
O
N
O
parallel helix, Hoogsteen base pair
H H
H
Major groove O
N H
N N
CH3
H N
N
H N
N O
H
antiparallel A helix Watson Crick base pair 5' - TTTTTTTTTT- 3' 5' - AAAAAAAAAA- 3' 3' - TTTTTTTTTT- 5'
WatsonCrick
Hoogsteen
H H
N
N
N H
O H
H
H O
N N
N H N N H H
WatsonCrick
protonated C required for base pairing H H N
H
N
H N
O
5' - CCCCCCCCCC- 3' ++++++++++ 5' - GGGGGGGGGG- 3' 3' - CCCCCCCCCC- 5'
Hoogsteen (+ indicates H +)
Polypurine Triplex Motif N N
N
anti parallel helix, Reverse Hoogsteen base pair
H N
N
H H
Major groove H N
N N
O
H H
N
CH3
N
H N
N O
H
antiparallel A helix Watson Crick base pair 3'-AAAAAAAAAA-5' 5'-AAAAAAAAAA-3' 3'-TTTTTTTTTT-5'
WatsonCrick
Reverse Hoogsteen
N N
N
O
H
H H
H
N
N
O
N N
N H N N H H
WatsonCrick
H H
N
H
N
H N
O
3'-GGGGGGGGGG-5' 5'-GGGGGGGGGG-3' 3'-CCCCCCCCCC-5'
Reverse Hoogsteen
Intramolecular Triplex NMR structure 1gn7.pdb
Intramolecular Triplex (1GN7.pdb) highlights
Yeast phenylalanine tRNA (4tna.pdb)
acceptor end triplex region
anticodon
Yeast phenylalanine tRNA (4tna.pdb)
acceptor end
anticodon
Base Pairing Found in tRNA Η
Ν CH3
Η Ν
Ο
Ν
CH3
Η
Ν
Ν
Ν
Ο Ο
Ν
Ν
Ν Η
Ν
Ν
Ο
T54
Η
m 1A58
Η Ν Ν
Ο
Ν Η
U69
G4 Ο
Ο Ν
Η
Ν Ν
Ν
Ν
Ν
Ν Η
Η
G18
Ν
Ο H
Ν
N
CH3 Ν
Ν
A9
Ν
Η Ν
Ν
Ν
Ν Ν
Ν
Ο
Ν
Ο Η Ν
Ν Η
U12
Η Η Ν Ν Ν
Ν Ν Η
Ο
A23
Η
Η
Η
Η
Η Ν Η
Ν
m 7G46 Ν
Ν
Ο
Η Ν
Η
C48 Ν
Ν
Η
Ν Ν
Η
O
Ν Ν
Ν
G15
H
ψ55
Ν
Ο
Η
Η
G22
Ο
C13
Chemical Probe Assays of Nucleic Acid Structure and Interactions Hyper reactive sites
Accessible sites *P
*P
*P
*P
*P
induce cleavage
induce cleavage *P
*P
*P
A
B
C
D
A. B. C. D.
- binder + binder pre conformational change post conformational change
Mechanism of strand cleavage following glycosidic bond hydrolysis
Base
B
hydrolysis
O O O
O P
-
O
O P
enolization
O
H O
H
O P
O
O
O
O P
-
O P
O
O
O
β-elimination (retro-Michael Rxn)
O P
H H O O
-O
H
O
B
O
H O
O
P
O
O
B
O P
O -
O
O
O H H O
O
O -
-
H
β-elimination (retro-Michael Rxn)
O
O
H
H
-
-
O
O-
O
O
hemiacetal abasic site
O
-
O
O
B
H
O
O P
O -
O
H
O
O
O P
O
-
O
O P -
-O
O P
O
O
O
3'-phosphate
5'-phosphate
In the presence of piperidine, the β-elimination reactions may take place through the enamine.
-
Dimethyl Sulfate Probe for the Accessibility of N7. A Major Groove Accessibility Probe. CH 3
O O S O CH 3 O
CH 3
N
N
N
DMS
N
G>A
Dimethyl sulfate (DMS). Alkylates sterically accessible N7 of purines. (See the Maxam-Gilbert G reaction in chapter on sequencing.) Reactivity: 1. The N7 of G in single strand and duplex DNA. 2. The N7 of purines in the anti conformation.
Diethylpyrodicarbonate (DEPC) probe of single stranded purines O O CH3
O
O
O O
O
CH3 +
N
N
N
N
DEPC A or G Approach is from the major groove side. DEPC is bulky, and reaction with N7 is inhibited in duplex DNA. Reactive N7: 1. Single strand DNA. 2. Loops of cruciforms. 3. Purines in the syn conformation in Z DNA.
Osmium tetroxide and KMnO4 oxidation of 5,6-double bond of pyrimidines O H O
CH3 O N O
N
H
Os N
H
O O N
N
O
O CH 3 O N O Os N
H
O
O
N
Reagent must approach from above or below the plane of the pyrimidine therefore it will not work well on stacked DNA Reactivity: 1. Reacts with T's at junctions between B & Z DNA. 2. Reacts with T's in cruciform loops.
Hydroxylamine reactions with C HO
NH2 N N
HO
N N
O
N
N
H O
N HO N
N
H O
H
labile to piperidine
Reagent must approach from above or below the plane of the pyrimidine therefore it will not work well on stacked DNA Reactivity: 1. Reacts with C's at junctions betwee B & Z DNA. 2. Reacts with C's at junctions between out of phase Z DNA blocks such as the sequence shown below: 5'-GCGCGC-CGCGCG-3' 3'-CGCGCG-GCGCGC-5' 3. Reacts with C's in cruciform loops.
H DNA Structure 5'
3'
single strand GAAGGA
triplex
5'3'-
5'3'-
CTTCCT GAAGGA CTTCCT
AGGAAG TCCTTC
GAAGGA CTTCCT
TCCTTC AGGAAG TCCTTC
triplex
AGGAAG
5' 3'
single strand
5' 3'
3' 5'
Chemical Probing of H-DNA Johnston, B.H. (1988) The S1-sensitive form of d(CT)n.d(A-G)n: chemical evidence for a three-stranded structure in plasmids. Science, 241, 1800-4.
a) normal superhelical density b) Higher superhelical density (higher torsional stress)
The End Replication Problem
Succesive rounds of replication lead to progressive shortening of the ends of DNA
Telomerase solves the End Replication Problem
ribonucleoprotein
elongation
translocation
Schematic structure of a telomere
Annu. Rev. Pharmacol. Toxicol. 2003. 43:359–79
The G’s in the telomere sequence can form Quartets via Hoogsteen Base Pairing,
• Hoogsteen base pairing leads to parallel strands if glycosyl bonds are anti • Center of quartet has large negative electrostatic potential H
H N
N
N
N
H
N H
N H
H N
N H N
N
N
+ O
N
N
O
H
H
N H
O
N
H N
H
H
N H H
N
O N
N H
Four possible orientations of Gn strands
parallel
antiparallel
mixed (3+1)
antiparallel
Ways of forming intramolecular quadruplex formation with [GxNy]z with 3 types of loops: propeller, lateral, diagonal propeller
hybrid
3'
lateral loop
3'
external or propeller loop
external loop
5' 5' lateral loop
chair
lateral loop
basket
lateral loop
lateral loop
lateral loop
3' 3' 5'
5' lateral loop
diagonal loop
Glycosyl conformation depends on strand orientation. Bases in one base quad can all flip from anti to syn, and syn to anti a
a
a
a
s
a
a
a
a
a
s
a a
a
s
s
a
s
s
a
a
a
a
a
Flip central Base quad s
a a
s
a
s
s
a
a
s
s
a
a a
s a
s
s
a
s
a
s
a
s
Front. Chem. 4:38. doi: 10.3389/fchem.2016.00038
J. Phys. Chem. B, Vol. 110, No. 32, 2006 16077
Characterized human telomeric DNA Gquadruplex structures lateral 3
1
T20
T8
A3 A21 G4
syn A9
G10
T8
G11
G17
G6 T7
G12
G16
G23
G12
T20
G22
G4 T13
A15
T13
T14
2
A3
2
lateral
hybrid-1 lateral
T8
T18
T7
Na
G10
T8 T20
T19
A9
G6
G10
syn
G6 G11
Na+ G17
T7 A21
+
G18
diagonal
lateral
A9
A21
syn
T14
hybrid-2 lateral
T19
G11
G5
A21 A15
G6
T19
G5
1
G10
syn
anti
G24
propellar
anti
G18
T7
3
T19
A9
anti
G18
K
+
G17
G5
G5
G11
G4 G12
G12
Na+ G16
G16
G4 T13
A15
X Y A15
5'
3' T14
T14
diagonal
basket
diagonal
form 3
T13
Folding and Unfolding Pathways of the Human Telomeric G-Quadruplex
J. Mol. Biol. (2014) 426, 1629–1650
Chemical Probes of G-quartet structures. Hoogsteen base pairing interferes with G reaction (reaction at N7).
PNAS 2002 99 11593-11598