PROPERTIES OF DNA I PRIMARY SEQUENCE A. Base Pairing B. Nucleotide Conformations C. Types of DNA D. Supercoiling E. Restriction nucleases F. DNA Sequencing

Lactam (keto) Lactim (enol) TAUTOMERIC FORMS

Conformational Rules Nucleotides are flexible and can be twisted about their C-O-P bonds There are 7 torsion angles in a nucleotide One torsion angle joins base to sugar The deoxyribose ring is “puckered” and not flat Puckering influences position of PO4 on the 3’ and 5’ position of the ring

CONFORMATION OF SUGAR-PHOSPHATE Nucleotides in DNA have 7 torsion angles that govern orientation of nucleotide chain. Favorable Unfavorable Z-DNA

Torsion Angles in a Nucleotide Rotation at  hindered C-O-P bonds flexible C-C, C-O, P-O flexible

Conformations of the Deoxyribose Ring No Tilt Tilts Bases C2’-endo 5’ C3’-endo C3’-endo is found in A-DNA C2’-endo is found in B-DNA Note 3’ and 5’ PO4 group in C2’endogives bases an arrangement more perpendicular to the helix axis

TYPES OF DNA 1. 3 types: A, B, and Z 2. Not in equilibrium 3. Transition depends on humidity, temperature and DNA binding proteins

B-DNA (Watson-Crick) 90% humidity 1. Two Antiparallel polynucleotide strands 2. Sugar phosphates on periphery (Minimize charge repulsion) 3. Helix approximately 20 Angstroms in diameter 4. 10.5 base pairs per turn, ~36 degrees per base pair 5. Bases flat, perpendicular to axis 6. Major and minor grooves readily apparent

Major Minor

A DNA: What distinguishes A DNA from B DNA? A DNA is wider and flatter: 11 base-pairs per turn instead of 10.5. The helix diameter is 26 angstroms instead of 20. The major groove is narrow and subdued. Is base-pairing the same? Yes. But the bases join around the axis and not through the axis and are tilted 20 degrees. Why is A DNA important to know? A DNA is seen in single-stranded RNA molecules that fold back on themselves. A DNA is also seen in DNA-RNA hybrids. Low humidity causes it to form from B DNA

Z-DNA 1. Left handed helix 2. 18 Angstron diameter 3. No major groove 4. 12 base pairs per turn 5. Repeating units is a dinucleotide dRY or dYR: d(GC) d(CG) d(AC) d(GT) 6. Formation also depends on high salt to block charge repulsion

Z-DNA Transition region CGCGCG Negative twisted DNA

DNA Dialogue What forces hold a typical DNA molecule together? ANS: Hydrogen bonds between bases either through or or around the axis and base stacking What is base stacking? Stacking implies vertical interactions between bases as they sit on top of one another What sort of interactions? Mainly van der Waal forces created by hydrophobic interactions Are the forces of interaction the same for all bases? No. Stacking interactions between G and C give rise to greater stacking energy than A to T

What does this do to the DNA? Ans: The greater the GC content of DNA the greater the stability, thermal stability in particular What do you mean by thermal stability? Two ways to view thermal stability. It could be the heat energy required to separate or melt the strands What else besides heat? Thermal could reflect the strength of bonding of the two DNA strands to one another though a combination of both H-bonding and base stacking How is thermal stability measured? Next slide

A260 Melting Point of DNA Temperature oC Tm (melting temperature) Lower G + C Higher G + C A260 Temperature oC 50 70 Tm (melting temperature) 90 Hyperchromicity

Examples: DNA-Protein Interactions Rule: The interaction of proteins with nucleic acids is an important biological property governing nucleic acid function in replication and transcription Examples: 1. Endo and Exonucleases, kinases, ligases 2. Histones 3. Transcription Repressor proteins 4. Transcription Enhancer proteins 5. Topoisomerases 6. Single strand DNA binding proteins 7. DNA-RNA polymerases

Rule: In DNA-RNA-Protein interactions there must exist a structural harmony between the nucleic acid and the protein at points of contact Transcription Factors 1. Helix-turn-helix protein (HTH) a. Bind to operators b. Typically dimeric c. Repress transcription of specific genes 2. Zinc finger protein a. Eukaryotic b. Cysteine and Histidine rich 1. Typically cys2-his2

Zinc Fingers (continued) c. Designed to recognize asymmetric base sequences 3. Leucine Zippers a. Leucine repeats every 7th residue b. Dimerizes as a coil-coil, -leucine are teeth c. Basic region adjacent to zipper binds to DNA d. Differ from other transcription factors by engaging DNA at basic end of protein e. Typical is the AP1 transcription factor composed of c-jun and c-fos leucine zippers, related to v-jun and v-fos, known heterodimeric oncogenes

Y shape is a typical feature of zippers bZIP Zinc Finger Project into DNA grooves Y shape is a typical feature of zippers bZIP Zinc Finger DNA must have an inverted repeat to accommodate bZIP Leucine Zipper