1. DNA STRUCTURE STRUCTURE, FORCES AND TOPOLOGY

2. 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

3. 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

4. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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?

14. 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

15. 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

16. FORCES THAT STABILIZE NUCLEIC ACID STRUCTURES SUGAR-PHOSPHATE CHAIN CONFORMATIONS BASE PAIRING BASE-STACKING,HYDROPHOBIC IONIC INTERACTIONS

17. 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

18. 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

19. 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

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

21. 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

22. 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-

23. 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

24. 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 T m OF DS- DNA AS [CATION] INCREASES? WHY?

25. IONIC INTERACTIONS DIVALENT CATIONS ARE GOOD SHIELDING AGENTS MONOVALENT CATIONS INTERACT NON-SPECIFICALLY – FOR EXAMPLE, IN AFFECTING T m 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

26. 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

27. 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

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

29. 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

30. 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

31. 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”?