Introduction to Molecular and Cell Biology, Biol. 220 Lecture 6: The Structure of nucleic acids
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  "DNA, the stuff genes are made of!"

What are the building blocks of DNA?

Sugars:

Fig. 1Ribose and 2'-deoxyribose are the basic sugars for RNA and DNA

Phosphates:

Fig. 2 Phosphate group

Bases composed of Purines and Pyrimidines:

Fig. 3 Purines

Fig. 4 Pyrimidines

How are these components linked together?

The nucleotides are composed as a central sugar linked to phosphates at the 5' carbon and a base at the 1' carbon.

Nucleotides are linked together through an alternating sugar - phosphate backbone.

Fig. 5 Single strand of DNA showing alternating sugar-phosphate backbone.

DNA is composed of two antiparallel strands held together by hydrogen bonding between the bases: G=C and A=T (or A=U in RNA).

Fig. 6 Base pairing between two antiparallel strands of DNA.

Questions:

1. What would be the sequence of the complementary strand of DNA for:

TAGACATGAGATAT

2. Using "A,C,G and T" to represent the bases, and using "P" and "S" for the phosphate and the sugar respectively, draw the double-stranded DNA structure for the sequence ACGT. Label the 5' and 3' ends of each strand. Use solid lines to represent covalent bonds between the sugar, base and phosphate. Use a dotted line to represent hydrogen bonds.

What is the Structure of DNA?

DNA usually forms a right-handed helix.

Fig. 7 B-form and A-form of DNA (right handed)

But when the alternating ...GCGCGC... is found the DNA assumes a zig zag left-handed helix.

Fig. 9 Z-form of DNA, (left handed)

Compare the three forms side by side:

Fig. 10 Comparison of A-, B-, and Z-forms of DNA

Fig. 4.6  Comparison of different DNA forms.

How stable is DNA?

The covalent bonds that link the nucleotides together are quite stable, however, since the antiparallel strands are held together by Hydrogen-bonds, they are subject to becoming separated from each other:

When complementary DNA strands are "base-paired" with each other through hydrogen bonds they are said to be in their "native" state.

When the bonds between the complementary strands are broken and the strands separate, the DNA is said to be denatured (not degraded).

When the denatured complementary strands come back together and assume a native state they have "reannealed" or "renatured".

Fig. 4.8  Denaturation by heat or pH > 11.3, and renaturation.

How can we tell if DNA is in a native or denatured state?

DNA absorbs maximally at 260 nm (in the UV range, Visible light 400-700 nm)

Proteins absorb maximally at 280 nm

The ability of DNA to absorb light (260nm, UV) increases as denaturation progresses. Absorbance is primarily a property of the nitrogenous bases. When they are hydrogen bonded and in close proximity they cannot absorb light as well as when they are exposed.

Absorbance goes up at 260nm even higher when the DNA is completely hydrolysed (broken down into single nulcleotides).

Fig. 4.9a  Melting curve for double and single stranded DNA.

Question: Which DNA sequence will have the highest Tm?

ATTATTACGT      or     GCGAGAGTGC
  TAATAATGCA             CGCTCTCACG

We can also observe DNA structure under the electron microscope.

Fig. 4.11  Supercoiled (Form I) and relaxed (Form II) closed circular DNA.

Questions:

1. What impact does supercoiling have on a DNA structure?

2. Can a linear piece of DNA experience supercoiling?

What impact does pH have on DNA structure?

Hydrogen bonding is very sensitive to H+ concentration

 At pH greater than 11.3 (alkaline conditions) all hydrogen bonds are eliminated and DNA is completely denatured (in single stranded form).

 Acid conditions also denature DNA but... there is damage to phosphodiester linkages. Phosphodiester bonds are resistant to alkaline pH so use of alkaline pH is method of choice for deliberately denaturing DNA in the laboratory.

So why does dna denature when all of the salt is removed?

 Interestingly, negative "-" repulsive forces also influence the DNA double helix.

Recall, the negatively charged phosphate groups that make up the DNA backbone.

Charges are usually neutralized by bound + ions such as Na+, K+, and Mg++.

*In pure water the "-" repulsions are so great that the strands seprate or denaturate.

Question: So, what three conditions need to be controlled to maintain DNA as a double-stranded molecule?

How are the covalent bonds in DNA formed and cut?

The P-P that is released during synthesis is further broken down into two inorganic phosphates.

DNA is broken down by the action of exonucleases, which chew bases off from the ends, or endonucleases, which break bonds between the ribose and phosphate in the interior of the DNA.

Fig. 1 Nuclease Action

Restriction Endonucleases

Some endonucleases recognize specific base sequences between 4 and 8 base pairs in length.

This class of endonuclease is very critical to the technique of genetic engineering.
Type II Restriction Endonucleases recognize a palindromic sequence (reads the same forward as backwards) and make staggered cuts.

 For example, the EcoRI endonuclease recognizes the palindromic sequence:

     5'------G-A-A-T-T-C-----3'
     3'------C-T-T-A-A-G-----5'

Note that reading this sequence on either the top or bottom strands from the 5' to 3' ends it reads in the same order (palindromic).

The enzyme will cut the phosphodiester bond between G-A on both strands, resulting in a staggered double-stranded cut of the DNA.
 

     5'------G                     A-A-T-T-C-----3'
     3'------C-T-T-A-A                     G-----5'
 

Restriction Enzymes and Genetic Engineering

This is useful in genetic engineering because the overhangs can be hybridized to like cut DNA from another source and a fragment inserted at the original restriction enzyme site:
 

                                               |
                                              V

5'--G A-A-T-T-C---insert-G A-A-T-T-C--3'
3'--C-T-T-A-A G---insert-C-T-T-A-A G--5'

Enzymes called ligases can reform the phosphodiester bonds -:

5'--G-A-A-T-T-C---insert-G-A-A-T-T-C--3'
3'--C-T-T-A-A-G---insert-C-T-T-A-A-G--5'

How can DNA breathe if it has no lungs?

Double stranded state is stable, but double stranded regions frequently open to become temporary single-stranded bubbles (breathing).

 In AT rich areas, breathing is much more common. (Remember this for later sections.)

Question: In DNA replication and transcription, the DNA must denature in a localized region so that replication and transcription enzymes can begin making copies (we will study both of these processes later in the semester). Would you expect the sites on the DNA where these enzymes bind to be AT-rich or GC-rich?

A Story

When I was a kid, I loved to climb trees. I had a treehouse and would spend long summer days enhancing and enjoying my castle in the sky. I spent so much time up in the trees that my father could often times be heard calling up into the trees "Come down you little monkey...."

So, now as a biology 220 student you can test my father's theory that I am a monkey.

How can you do that? Well, let's look at what you know.

You know that DNA is genetic material.

You know that you can get the strands of DNA to separate when heated.

You know that these strands can be renatured back together under the proper conditions.

Only when the bases match between the two strands will they form a stable complex.

We can look at the structure of DNA under an electron microscope.

We can follow the % of single stranded vs double stranded DNA during renaturation with spectrophotometry. (ssDNA absorbs 37% more than dsDNA).

What would happen if you took DNA from me and a monkey, denatured the DNA, mixed it together and then allowed it to reanneal?

Consider what what you would see under the EM if there were matches or lack of matches between the two sources of DNA.

Humm.......

Fig. 1 Hybridization and Heteroduplex Analysis

What if we followed the rate of renaturation in a spectrophometer. How could you determine how related the two DNAs are?

Fig. 3 Renaturation Curve with unique sequences.

What if we cut my DNA and the Monkeys DNA with a restriction enzyme and then compared the sizes of the fragments that were generated?

Are there some other ways to compare the DNAs?

 

Created 2004 by CA Rinehart• email CA Rinehart

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