Download DNA STRUCTURE

DNA STRUCTURE
STRUCTURE, FORCES
AND TOPOLOGY
DNA GEOMETRY
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A POLYMER OF DEOXYRIBONUCLEOTIDES
DOUBLE-STRANDED
INDIVIDUAL deoxyNUCLEOSIDE TRIPHOSPHATES ARE
COUPLED BY PHOSPHODIESTER BONDS
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ESTERIFICATION
LINK 3’ CARBON OF ONE RIBOSE WITH 5’ C OF ANOTHER
TERMINAL ENDS : 5’ AND 3’
A “DOUBLE HELICAL” STRUCTURE
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COMMON AXIS FOR BOTH HELICES
“HANDEDNESS” OF HELICES
ANTIPARALLEL RELATIONSHIP BETWEEN 2 DNA STRANDS
DNA GEOMETRY
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PERIPHERY OF DNA
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SUGAR-PHOSPHATE CHAINS
CORE OF DNA
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BASES ARE STACKED IN PARALLEL FASHION
CHARGAFF’S RULES
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A=T
G=C
“COMPLEMENTARY” BASE-PAIRING
TAUTOMERIC FORMS OF BASES
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TWO POSSIBILITIES
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KETO (LACTAM)
ENOL (LACTIM)
PROTON SHIFTS BETWEEN TWO FORMS
IMPORTANT IN ORDER TO SPECIFY HYDROGEN
BONDING RELATIONSHIPS
THE KETO FORM PREDOMINATES
MAJOR AND MINOR GROOVES
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MINOR
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MAJOR
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EXPOSES EDGE FROM WHICH C1’ ATOMS EXTEND
EXPOSES OPPOSITE EDGE OF BASE PAIR
THE PATTERN OF H-BOND POSSIBILITIES IS
MORE SPECIFIC AND MORE DISCRIMINATING IN
THE MAJOR GROOVE
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STUDY QUESTION: LOCATE ALL OF THE POSSIBILITIES
FOR H-BONDING IN THE MAJOR AND MINOR
GROOVES FOR THE 4 POSSIBLE BASE-PAIRS
STRUCTURE OF THE DOUBLE HELIX
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THREE MAJOR FORMS
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B-DNA
A-DNA
Z-DNA
B-DNA IS BIOLOGICALLY THE MOST COMMON
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RIGHT-HANDED (20 ANGSTROM (A) DIAMETER)
COMPLEMENTARY BASE-PAIRING (WATSON-CRICK)
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A-T
G-C
EACH BASE PAIR HAS ~ THE SAME WIDTH
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10.85 A FROM C1’ TO C1’
A-T AND G-C PAIRS ARE INTERCHANGEABLE
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“PSEUDO-DYAD” AXIS OF SYMMETRY
GEOMETRY OF B-DNA
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IDEAL B-DNA HAS 10 BASE PAIRS PER TURN
BASE THICKNESS
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AROMATIC RINGS WITH 3.4 A THICKNESS TO RINGS
PITCH = 10 X 3.4 = 34 A PER COMPLETE TURN
AXIS PASSES THROUGH MIDDLE OF EACH BP
MINOR GROOVE IS NARROW
MAJOR GROOVE IS WIDE
IN CLASS EXERCISE: EXPLORE THE
STRUCTURE OF B-DNA. PAY SPECIAL
ATTENTION TO THE MAJOR, MINOR GROOVES
A-DNA
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RIGHT-HANDED HELIX
WIDER AND FLATTER THAN B-DNA
11.6 BP PER TURN
PITCH OF 34 A
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BASE PLANES ARE TILTED 20 DEGREES WITH RESPECT
TO HELICAL AXIS
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 AN AXIAL HOLE
HELIX AXIS PASSES “ABOVE” MAJOR GROOVE
 DEEP MAJOR AND SHALLOW MINOR GROOVE
OBSERVED UNDER DEHYDRATING CONDITIONS
A-DNA
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WHEN RELATIVE HUMIDITY IS ~ 75%
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B-DNA  A-DNA (REVERSIBLE)
MOST SELF-COMPLEMENTARY OLIGONUCLEOTIDES OF < 10 bp CRYSTALLIZE IN A-DNA CONF.
A-DNA HAS BEEN OBSERVED IN 2 CONTEXTS:
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AT ACTIVE SITE OF DNA POLYMERASE (~ 3 bp )
GRAM (+) BACTERIA UNDERGOING SPORULATION
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SASPs INDUCE B-DNA TO  A-DNA
RESISTANT TO UV-INDUCED DAMAGE
– CROSS-LINKING OF PYRIMIDINE BASES
Z-DNA
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A LEFT-HANDED HELIX
SEEN IN CONDITIONS OF HIGH SALT CONCENTRATIONS
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IN COMPLEMENTARY POLYNUCLEOTIDES WITH
ALTERNATING PURINES AND PYRIMIDINES
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REDUCES REPULSIONS BETWEEN CLOSEST PHOSPHATE
GROUPS ON OPPOSITE STRANDS (8 A VS 12 A IN B-DNA)
POLY d(GC) · POLY d(GC)
POLY d(AC)  POLY d(GT)
MIGHT ALSO BE SEEN IN DNA SEGMENTS WITH ABOVE
CHARACTERISTICS
Z-DNA
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12 W-C BASE PAIRS PER TURN
A PITCH OF 44 DEGREES
A DEEP MINOR GROOVE
NO DISCERNIBLE MAJOR GROOVE
REVERSIBLE CHANGE FROM B-DNA TO Z-DNA
IN LOCALIZED REGIONS MAY ACT AS A
“SWITCH” TO REGULATE GENE EXPRESSION
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? TRANSIENT FORMATION BEHIND ACTIVELY TRANSCRIBING RNA POLYMERASE
STRUCTURAL VARIANTS OF DNA
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DEPEND UPON:
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SOLVENT COMPOSITION
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WATER
IONS
BASE COMPOSITION
IN-CLASS QUESTION: WHAT FORM OF
DNA WOULD YOU EXPECT TO SEE IN
DESSICATED BRINE SHRIMP EGGS?
WHY?
RNA
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UNLIKE DNA, RNA IS SYNTHESIZED AS A SINGLE STRAND
THERE ARE DOUBLE-STRANDED RNA STRUCTURES
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RNA CAN FOLD BACK ON ITSELF
DEPENDS ON BASE SEQUENCE
GIVES STEM (DOUBLE-STRAND) AND LOOP (SINGLESTRAND STRUCTURES)
DS RNA HAS AN A-LIKE CONFORMATION
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STERIC CLASHES BETWEEN 2’-OH GROUPS PREVENT THE
B-LIKE CONFORMATION
HYBRID DNA-RNA STRUCTURES
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THESE ASSUME THE A-LIKE CONFORMATION
USUALLY SHORT SEQUENCES
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EXAMPLES:
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DNA SYNTHESIS IS INITIATED BY RNA “PRIMERS”
DNA IS THE TEMPLATE FOR TRANSCRIPTION TO RNA
FORCES THAT STABILIZE NUCLEIC
ACID STRUCTURES
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SUGAR-PHOSPHATE CHAIN CONFORMATIONS
BASE PAIRING
BASE-STACKING,HYDROPHOBIC
IONIC INTERACTIONS
SUGAR-PHOSPHATE CHAIN IS
FLEXIBLE TO AN EXTENT
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CONFORMATIONAL FLEXIBILITY IS
CONSTRAINED BY:
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SIX TORSION ANGLES OF SUGAR-PHOSPHATE
BACKBONE
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TORSION ANGLES AROUND N-GLYCOSIDIC BOND
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RIBOSE RING PUCKER
TORSION ANGLES
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SIX OF THEM
GREATLY RESTRICTED RANGE OF ALLOWABLE
VALUES
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STERIC INTERFERENCE BETWEEN RESIDUES IN
POLYNUCLEOTIDES
ELECTROSTATIC INTERACTIONS OF PHOS. GROUPS
A SINGLE STRAND OF DNA ASSUMES A
RANDOM COIL CONFIGURATION
THE N-GLYCOSIDIC TORSION ANGLE
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TWO POSSIBILITIES, STERICALLY
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SYN
ANTI
PYRIMIDINES
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ONLY ANTI IS ALLOWED
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STERIC INTERFERENCE BETWEEN RIBOSE AND THE C2’
SUBSTITUENT OF PYRIMIDINE
PURINES
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CAN BE SYN OR ANTI
IN MOST DOUBLE-HELICAL STRUCTURES,
ALL BASES IN ANTI FORM
GLYCOSIDIC TORSION ANGLES IN
Z-DNA
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ALTERNATING
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PYRIMIDINE: ANTI
PURINE: SYN
WHAT HAPPENS WHEN B-DNA SWITCHES TO Z-DNA?
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THE PURINE BASES ROTATE AROUND GLYCOSIDIC BOND
FROM ANTI TO SYN
THE SUGARS ROTATE IN THE PYRIMIDINES
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THIS MAINTAINS THE ANTI CONFORMATIONS
RIBOSE RING PUCKER
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THE RING IS NOT FLAT
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CROWDING IS RELIEVED BY PUCKERING
TWO POSSIBILITIES FOR EACH OF C2’ OR C3’:
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SUBSTITUENTS ARE ECLIPSED IF FLAT
ENDO: OUT-OF-PLANE ATOM ON SAME SIDE OF RING AS C5’
EXO; DISPLACED TO OPPOSITE SIDE
C2’ ENDO IS MOST COMMON
CAN ALSO SEE C3’-ENDO AND C3’-EXO
LOOK AT RELATIONSHIPS BETWEEN THE PHOSPHATES:
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IN C3’ ENDO- THE PHOSPHATES ARE CLOSER THAN IN C2’
ENDO-
RIBOSE RING PUCKER
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B-DNA HAS THE C2’-ENDO-FORM
A-DNA IS C3’-ENDO
Z-DNA
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PURINES ARE ALL C3’-ENDO
PYRIMIDINES ARE ALL C2’-ENDO
CONCLUSION: THE RIBOSE PUCKER GOVERNS
RELATIVE ORIENTATIONS OF PHOSPHATE
GROUPS TO EACH SUGAR RESIDUE
IONIC INTERACTIONS
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THE DOUBLE HELIX IS ANIONIC
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DOUBLE-STRANDED DNA HAS HIGHER ANIONIC
CHARGE DENSITY THAT SS-DNA
THERE IS AN EQUILIBRIUM BETWEEN SS-DNA
AND DS-DNA IN AQUEOUS SOLUTION:
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MULTIPLE PHOSPHATE GROUPS
DS-DNA == SS-DNA
QUESTION: WHAT HAPPENS TO THE Tm OF DSDNA AS [CATION] INCREASES? WHY?
IONIC INTERACTIONS
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DIVALENT CATIONS ARE GOOD SHIELDING AGENTS
MONOVALENT CATIONS INTERACT NON-SPECIFICALLY
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DIVALENT INTERACT SPECIFICALLY
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FOR EXAMPLE, IN AFFECTING Tm
BIND TO PHOSPHATE GROUPS
MAGNESIUM (2+) ION
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STABILIZES DNA AND RNA STRUCTURES
ENZYMES THAT ARE INVOLVED IN RXNS’ WITH NUCLEIC
ACID USUALLY REQUIRE Mg(2+) IONS FOR ACTIVITY
BASE STACKING
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PARTIAL OVERLAP OF PURINE AND PYRIMIDINE
BASES
IN SOLID-STATE (CRYSTAL)
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VANDERWAALS FORCES
IN AQUEOUS SOLUTION
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MOSTLY HYDROPHOBIC FORCES
ENTHALPICALLY-DRIVEN
ENTROPICALLY-OPPOSED
OPPOSITE TO THAT OF PROTEINS
BASE-PAIRING
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WATSON-CRICK GEOMETRY
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THE A-T PAIRS USE ADENINE’S N1 AS THE H-BOND
ACCEPTOR
HOOGSTEEN GEOMETRY
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N7 IS THE ACCEPTOR
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IN DOUBLE HELICES, W-C IS MORE STABLE
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SEEN IN CRYSTALS OF MONOMERIC A-T BASE PAIRS
ALTHOUGH HOOGSTEIN IS MORE STABLE FOR A-T PAIRS, WC IS MORE STABLE IN DOUBLE HELICES
CO-CRYSTALLIZED MONOMERIC G-C PAIRS
ALWAYS FOLLOW W-C GEOMETRY
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THREE H-BONDS
HYDROGEN BONDING
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REQUIRED FOR SPECIFICITY OF BASE PAIRING
NOT VERY IMPORTANT IN DNA STABILIZATION
HYDROPHOBIC FORCES ARE THE MOST IMPT.’
THE TOPOLOGY OF DNA
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“SUPERCOILING” : DNA’S “TERTIARY STRUCTURE
L = “LINKING NUMBER”
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A TOPOLOGIC INVARIANT
THE # OF TIMES ONE DNA STRAND WINDS AROUND THE
OTHER
L=T+W
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T IS THE “TWIST
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THE # OF COMPLETE REVOLUTIONS THAT ONE DNA STRAND
MAKES AROUND THE DUPLEX AXIS
W IS THE “WRITHE”
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THE # OF TIMES THE DUPLEX AXIS TURNS AROUND THE
SUPERHELICAL AXIS
DNA TOPOLOGY
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THE TOPOLOGICAL PROPERTIES OF DNA HELP
US TO EXPLAIN
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DNA COMPACTING IN THE NUCLEUS
UNWINDING OF DNA AT THE REPLICATION FORK
FORMATION AND MAINTENANCE OF THE
TRANSCRIPTION BUBBLE
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MANAGING THE SUPERCOILING IN THE ADVANCING
TRANSCRIPTION BUBBLE
DNA TOPOLOGY
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AFTER COMPLETING THE 13 IN-CLASS EXERCISES, TRY
TO ANSWER THE FOLLOWING QUESTIONS:
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(1) THE HELIX AXIS OF A CLOSED CIRCULAR DUPLEX DNA IS
CONSTRAINED TO LIE IN A PLANE. THERE ARE 2340 BASE PAIRS IN THIS
PIECE OF DNA AND, WHEN CONSTRAINED TO THE PLANE, THE TWIST IS
212.
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DETERMINE “L”, “W” AND “T” FOR THE CONSTRAINED AND UNCONSTRAINED
FORM OF THIS DNA.
(2) A CLOSED CIRCULAR DUPLEX DNA HAS A 100 BP SEGMENT OF
ALTERNATING C AND G RESIDUES. ON TRANSFER TO A SOLUTION WITH
A HIGH SALT CONCENTRATION, THE SEGMENT MAKES A TRANSITION
FROM THE B-FORM TO THE Z-FORM. WHAT IS THE ACCOMPANYING
CHANGE IN “L”, “W”. AND “T”?