properties of dna i primary sequence a. base pairing

PROPERTIES OF DNA I

B. Nucleotide Conformations

PRIMARY SEQUENCE

C. Types of DNAD. SupercoilingE. Restriction nucleasesF. DNA Sequencing

A. Base Pairing

Lactim(enol)

Adenine

Guanine

Cytosine

Uracil (Thymine)

HOH

(H,CH3)OH

N

N (H,CH3)

N

N N

NHNH

NH

N

N

O

H

O

N

N N

NNH2

N

NNH2 N

N

N

N

O

OH

O N

NNH2

ON

NNH2 N

N

imino

imino

amino

keto enol

enolketo

Lactam

TAUTOMERIC FORMS

(keto)

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

NH

H2

ON

N N

N

O

O OHH

HOCH2interaction

syn-Guanosine anti-Guanosine

HO

N

NN

N

NH2HOCH2

O

O OHH

Nucleotides in DNA have 7 torsion angles that govern orientation of nucleotide chain.

FavorableUnfavorable

Z-DNA

Torsion Angles in a Nucleotide

• 7 torsion angles• Rotation at hindered• C-O-P bonds flexible• C-C, C-O, P-O flexible

C3’-endo C2’-endo

Conformations of the Deoxyribose Ring

5’

C3’-endo is found in A-DNAC2’-endo is found in B-DNA

Note 3’ and 5’ PO4 group in C2’endogives bases an arrangement more perpendicular to the helix axis

Tilts Bases No Tilt

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 diameter4. 10.5 base pairs per turn, ~36 degrees per base pair

5. Bases flat, perpendicular to axis

6. Major and minor grooves readily apparent

B-DNA

MajorMinor

A DNA:

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

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?

But the bases join around the axis and not through the axis and are tilted 20 degrees. Yes.

Z-DNA

1. Left handed helix2. 18 Angstron diameter3. No major groove4. 12 base pairs per turn5. 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

Negative twisted DNA

CGCGCG

What forces hold a typical DNA molecule together?DNA Dialogue

ANS: Hydrogen bonds between bases either through oror around the axis and base stackingWhat is base stacking?Stacking implies vertical interactions between bases asthey sit on top of one anotherWhat sort of interactions?Mainly van der Waal forces created by hydrophobicinteractions Are the forces of interaction the same for all bases?No. Stacking interactions between G and C give rise togreater stacking energy than A to T

What does this do to the DNA?Ans: The greater the GC content of DNA the greaterthe stability, thermal stability in particular

What do you mean by thermal stability?

Two ways to view thermal stability. It could be the heatenergy required to separate or melt the strandsWhat else besides heat?

Thermal could reflect the strength of bonding of the twoDNA strands to one another though a combinationof both H-bonding and base stacking

How is thermal stability measured?Next slide

50 70 90

Hyperchromicity

A260

Tm (melting temperature)

Higher G + C

Lower G + C

Temperature oC

Melting Point of DNA

DNA-Protein Interactions

Rule: The interaction of proteins with nucleic acids is animportant biological property governing nucleic acidfunction in replication and transcription

Examples:

1. Endo and Exonucleases, kinases, ligases2. Histones3. Transcription Repressor proteins

5. Topoisomerases6. Single strand DNA binding proteins7. DNA-RNA polymerases

4. Transcription Enhancer proteins

Rule: In DNA-RNA-Protein interactions there must exist a structural harmony between the nucleic acid and the protein at points of contact

Transcription Factors1. Helix-turn-helix protein (HTH)

a. Bind to operatorsb. Typically dimericc. Repress transcription of specific genes

2. Zinc finger proteina. Eukaryoticb. Cysteine and Histidine rich

1. Typically cys2-his2

Zinc Fingers (continued)

c. Designed to recognize asymmetric base sequences

3. Leucine Zippersa. Leucine repeats every 7th residueb. Dimerizes as a coil-coil, -leucine are teethc. Basic region adjacent to zipper binds to DNAd. Differ from other transcription factors

by engaging DNA at basic end of proteine. 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

Zinc Finger

Leucine Zipper

Project intoDNA grooves

Y shape is a typicalfeature of zippers

DNA must have aninverted repeat toaccommodate bZIP

bZIP