DNA STRUCTURE

STRUCTURE, FORCES AND TOPOLOGY

DNA GEOMETRY

A POLYMER OF DEOXYRIBONUCLEOTIDES DOUBLE-STRANDED INDIVIDUAL deoxyNUCLEOSIDE TRIPHOSPHATES ARE

COUPLED BY PHOSPHODIESTER BONDS– ESTERIFICATION– LINK 3’ CARBON OF ONE RIBOSE WITH 5’ C OF ANOTHER– TERMINAL ENDS : 5’ AND 3’

A “DOUBLE HELICAL” STRUCTURE– COMMON AXIS FOR BOTH HELICES– “HANDEDNESS” OF HELICES– ANTIPARALLEL RELATIONSHIP BETWEEN 2 DNA STRANDS

DNA GEOMETRY

PERIPHERY OF DNA– SUGAR-PHOSPHATE CHAINS

CORE OF DNA– BASES ARE STACKED IN PARALLEL FASHION– CHARGAFF’S RULES

A = T G = C

– “COMPLEMENTARY” BASE-PAIRING

TAUTOMERIC FORMS OF BASES

TWO POSSIBILITIES– 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

MINOR– EXPOSES EDGE FROM WHICH C1’ ATOMS EXTEND

MAJOR– EXPOSES OPPOSITE EDGE OF BASE PAIR

THE PATTERN OF H-BOND POSSIBILITIES IS MORE SPECIFIC AND MORE DISCRIMINATING IN THE MAJOR GROOVE

– 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

THREE MAJOR FORMS– B-DNA– A-DNA– Z-DNA

B-DNA IS BIOLOGICALLY THE MOST COMMON– RIGHT-HANDED (20 ANGSTROM (A) DIAMETER)– COMPLEMENTARY BASE-PAIRING (WATSON-CRICK)

A-T G-C

– EACH BASE PAIR HAS ~ THE SAME WIDTH 10.85 A FROM C1’ TO C1’ A-T AND G-C PAIRS ARE INTERCHANGEABLE

– “PSEUDO-DYAD” AXIS OF SYMMETRY

GEOMETRY OF B-DNA

IDEAL B-DNA HAS 10 BASE PAIRS PER TURN BASE THICKNESS

– 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

RIGHT-HANDED HELIX WIDER AND FLATTER THAN B-DNA 11.6 BP PER TURN PITCH OF 34 A

AN AXIAL HOLE BASE PLANES ARE TILTED 20 DEGREES WITH RESPECT

TO HELICAL AXIS– HELIX AXIS PASSES “ABOVE” MAJOR GROOVE DEEP MAJOR AND SHALLOW MINOR GROOVE

OBSERVED UNDER DEHYDRATING CONDITIONS

A-DNA

WHEN RELATIVE HUMIDITY IS ~ 75%– B-DNA A-DNA (REVERSIBLE)

MOST SELF-COMPLEMENTARY OLIGONUCLEO- TIDES OF < 10 bp CRYSTALLIZE IN A-DNA CONF. A-DNA HAS BEEN OBSERVED IN 2 CONTEXTS:

– AT ACTIVE SITE OF DNA POLYMERASE (~ 3 bp )– GRAM (+) BACTERIA UNDERGOING SPORULATION

SASPs INDUCE B-DNA TO A-DNA RESISTANT TO UV-INDUCED DAMAGE

– CROSS-LINKING OF PYRIMIDINE BASES

Z-DNA

A LEFT-HANDED HELIX SEEN IN CONDITIONS OF HIGH SALT CONCENTRATIONS

– REDUCES REPULSIONS BETWEEN CLOSEST PHOSPHATE GROUPS ON OPPOSITE STRANDS (8 A VS 12 A IN B-DNA)

IN COMPLEMENTARY POLYNUCLEOTIDES WITH ALTERNATING PURINES AND PYRIMIDINES

– POLY d(GC) · POLY d(GC)– POLY d(AC) POLY d(GT)

MIGHT ALSO BE SEEN IN DNA SEGMENTS WITH ABOVE CHARACTERISTICS

Z-DNA

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

– ? TRANSIENT FORMATION BEHIND ACTIVELY TRAN-

SCRIBING RNA POLYMERASE

STRUCTURAL VARIANTS OF DNA

DEPEND UPON:– SOLVENT COMPOSITION

WATER IONS

– BASE COMPOSITION IN-CLASS QUESTION: WHAT FORM OF

DNA WOULD YOU EXPECT TO SEE IN DESSICATED BRINE SHRIMP EGGS? WHY?

RNA

UNLIKE DNA, RNA IS SYNTHESIZED AS A SINGLE STRAND THERE ARE DOUBLE-STRANDED RNA STRUCTURES

– RNA CAN FOLD BACK ON ITSELF– DEPENDS ON BASE SEQUENCE– GIVES STEM (DOUBLE-STRAND) AND LOOP (SINGLE-

STRAND STRUCTURES) DS RNA HAS AN A-LIKE CONFORMATION

– STERIC CLASHES BETWEEN 2’-OH GROUPS PREVENT THE B-LIKE CONFORMATION

HYBRID DNA-RNA STRUCTURES

THESE ASSUME THE A-LIKE CONFORMATION USUALLY SHORT SEQUENCES

EXAMPLES:

– DNA SYNTHESIS IS INITIATED BY RNA “PRIMERS”– DNA IS THE TEMPLATE FOR TRANSCRIPTION TO RNA

FORCES THAT STABILIZE NUCLEIC ACID STRUCTURES

SUGAR-PHOSPHATE CHAIN CONFORMATIONS BASE PAIRING BASE-STACKING,HYDROPHOBIC IONIC INTERACTIONS

SUGAR-PHOSPHATE CHAIN IS FLEXIBLE TO AN EXTENT

CONFORMATIONAL FLEXIBILITY IS CONSTRAINED BY:

– SIX TORSION ANGLES OF SUGAR-PHOSPHATE BACKBONE

– TORSION ANGLES AROUND N-GLYCOSIDIC BOND

– RIBOSE RING PUCKER

TORSION ANGLES

SIX OF THEM GREATLY RESTRICTED RANGE OF ALLOWABLE

VALUES– 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

TWO POSSIBILITIES, STERICALLY– SYN– ANTI

PYRIMIDINES– ONLY ANTI IS ALLOWED

STERIC INTERFERENCE BETWEEN RIBOSE AND THE C2’ SUBSTITUENT OF PYRIMIDINE

PURINES– CAN BE SYN OR ANTI

IN MOST DOUBLE-HELICAL STRUCTURES, ALL BASES IN ANTI FORM

GLYCOSIDIC TORSION ANGLES IN Z-DNA

ALTERNATING– PYRIMIDINE: ANTI– PURINE: SYN

WHAT HAPPENS WHEN B-DNA SWITCHES TO Z-DNA?– THE PURINE BASES ROTATE AROUND GLYCOSIDIC BOND

FROM ANTI TO SYN– THE SUGARS ROTATE IN THE PYRIMIDINES

THIS MAINTAINS THE ANTI CONFORMATIONS

RIBOSE RING PUCKER

THE RING IS NOT FLAT– SUBSTITUENTS ARE ECLIPSED IF FLAT

CROWDING IS RELIEVED BY PUCKERING TWO POSSIBILITIES FOR EACH OF C2’ OR C3’:

– 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:– IN C3’ ENDO- THE PHOSPHATES ARE CLOSER THAN IN C2’

ENDO-

RIBOSE RING PUCKER

B-DNA HAS THE C2’-ENDO-FORM A-DNA IS C3’-ENDO Z-DNA

– 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

THE DOUBLE HELIX IS ANIONIC– MULTIPLE PHOSPHATE GROUPS

DOUBLE-STRANDED DNA HAS HIGHER ANIONIC CHARGE DENSITY THAT SS-DNA

THERE IS AN EQUILIBRIUM BETWEEN SS-DNA AND DS-DNA IN AQUEOUS SOLUTION:

– DS-DNA == SS-DNA

QUESTION: WHAT HAPPENS TO THE Tm OF DS-DNA AS [CATION] INCREASES? WHY?

IONIC INTERACTIONS

DIVALENT CATIONS ARE GOOD SHIELDING AGENTS MONOVALENT CATIONS INTERACT NON-SPECIFICALLY

– FOR EXAMPLE, IN AFFECTING Tm

DIVALENT INTERACT SPECIFICALLY– BIND TO PHOSPHATE GROUPS

MAGNESIUM (2+) ION– STABILIZES DNA AND RNA STRUCTURES– ENZYMES THAT ARE INVOLVED IN RXNS’ WITH NUCLEIC

ACID USUALLY REQUIRE Mg(2+) IONS FOR ACTIVITY

BASE STACKING

PARTIAL OVERLAP OF PURINE AND PYRIMIDINE BASES

IN SOLID-STATE (CRYSTAL)– VANDERWAALS FORCES

IN AQUEOUS SOLUTION– MOSTLY HYDROPHOBIC FORCES– ENTHALPICALLY-DRIVEN– ENTROPICALLY-OPPOSED– OPPOSITE TO THAT OF PROTEINS

BASE-PAIRING

WATSON-CRICK GEOMETRY– THE A-T PAIRS USE ADENINE’S N1 AS THE H-BOND

ACCEPTOR HOOGSTEEN GEOMETRY

– N7 IS THE ACCEPTOR SEEN IN CRYSTALS OF MONOMERIC A-T BASE PAIRS

IN DOUBLE HELICES, W-C IS MORE STABLE– ALTHOUGH HOOGSTEIN IS MORE STABLE FOR A-T PAIRS,

W-C IS MORE STABLE IN DOUBLE HELICES CO-CRYSTALLIZED MONOMERIC G-C PAIRS

ALWAYS FOLLOW W-C GEOMETRY– THREE H-BONDS

HYDROGEN BONDING

REQUIRED FOR SPECIFICITY OF BASE PAIRING NOT VERY IMPORTANT IN DNA STABILIZATION HYDROPHOBIC FORCES ARE THE MOST IMPT.’

THE TOPOLOGY OF DNA

“SUPERCOILING” : DNA’S “TERTIARY STRUCTURE L = “LINKING NUMBER”

– A TOPOLOGIC INVARIANT– THE # OF TIMES ONE DNA STRAND WINDS AROUND THE

OTHER L = T + W

– T IS THE “TWIST THE # OF COMPLETE REVOLUTIONS THAT ONE DNA STRAND

MAKES AROUND THE DUPLEX AXIS– W IS THE “WRITHE”

THE # OF TIMES THE DUPLEX AXIS TURNS AROUND THE SUPERHELICAL AXIS

DNA TOPOLOGY

THE TOPOLOGICAL PROPERTIES OF DNA HELP US TO EXPLAIN

– DNA COMPACTING IN THE NUCLEUS– UNWINDING OF DNA AT THE REPLICATION FORK– FORMATION AND MAINTENANCE OF THE

TRANSCRIPTION BUBBLE MANAGING THE SUPERCOILING IN THE ADVANCING

TRANSCRIPTION BUBBLE

DNA TOPOLOGY

AFTER COMPLETING THE 13 IN-CLASS EXERCISES, TRY TO ANSWER THE FOLLOWING QUESTIONS:

(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.

– 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”?