DNA

Learning Objectives

  1. Know that DNA is the genetic material by explaining the classic experimental evidence that DNA encodes genetic information, starting with bacterial transformation
  2. Describe the key features of the Watson-Crick model of DNA structure, including double helix, base-pairing, antiparallel orientation of strands, and types of bonds
  3. Predict outcomes from different modes of DNA replication in the Meselson-Stahl experiment
  4. Describe the basic machinery and process of DNA replication and predict outcomes if some elements of that machinery were missing or nonfunctional
  5. Explain how the properties of DNA polymerase and the structure of DNA causes DNA to be replicated differently for the leading and lagging strands

Part 1: The classic experimental evidence that DNA encodes genetic information.

Frederick Griffith discovered bacterial transformation: bacteria can acquire genetic material from their environment

Griffith worked with two strains of Streptococcus pneumoniae. The S strain produced a polysaccharide capsule and formed smooth-looking colonies. The R strain could not produce the polysaccharide capsule and formed rough-looking colonies. The S strain was virulent and killed mice. The R strain was non-virulent when injected into mice. Griffith reported in 1928 that mixtures of heat-killed extracts of the S cells plus live R cells killed mice, although neither injected alone were virulent. Autopsies of the dead mice showed bacterial cells in the blood. When cultured, these formed smooth colonies. He concluded that something from the extract of heat-killed S cells had transformed some of the R cells into S cells.

Griffiths experiment showing transformation of live R cells into S cells. Image from Wikipedia by Madprime, CC-BY-SA

Avery, McLeod and McCarty showed that bacterial transformation is due to DNA

Oswald Avery, Colin McLeod and Maclyn McCarty painstaking purified the transforming substance and reported in their 1944 paper that it was DNA. Their purification procedure eliminated polysaccharides, and the transforming substance was unaffected by proteases or ribonuclease (enzymes that cleave proteins and RNAs), but was destroyed by deoxyribonuclease.  

Here is a video summary of the design and conclusions from these two sets of experiments:

 

Alfred Hershey and Martha Chase showed that bacteriophage DNA infects cells, not protein

T2 is a virus that infects E. coli cells; viruses that infect prokaryotic cells are called bacteriophage or phage.  Bacteriphages are composed of just proteins and a DNA molecule packaged inside the phage. Here is a video of bacteriophage T4 landing on the surface of an E. coli cell and injecting its genetic material into the cell:

 

Left: bacteriophage proteins are labeled with 35S; right: phage DNA is labeled with 32P. Labeled phage were incubated briefly with cells to allow attachment and injection of genetic material into the cells. A bar blender was used to shear off the attached phage shells from the cells. Centrifugation pelleted the cells, leaving the phage shells suspended in the supernatant fraction. Radioactive phosphorus pelleted with the cells, but radioactive sulfur remained in the supernatant. Image from wikipedia, by Thomasione, with GFDL or CC-BY-SA license.

Hershey & Chase radioactively labeled either bacteriophage proteins (using radioactive sulfur) or bacteriophage DNA (using radioactive phosphate), and showed that labeled DNA entered infected cells, whereas labeled protein stayed outside. “Blending” refers to the use of a bar blender to shear off the attached bacteriophage particles from the infected cells. This is how bar blenders became standard equipment in microbiology and molecular biology labs.

Here is a video that summarizes the design and conclusions of the Hershey-Chase experiment:

 

Part 2: The Watson-Crick model of DNA structure

Watson & Crick constructed a model of DNA structure that fits Franklin’s X-ray diffraction data & Chargaff’s rules

The biochemist Erwin Chargaff analyzed the base composition of DNA from a wide variety of species, and found that although the percentages of A, G, C and T varied from species to species, the following proportions were always seen:

A = T; G = C

Watson and Crick used Rosalind Franklin’s X-ray diffraction data on purified DNA to deduce that DNA has a helical structure. They were able to solve the structure of DNA when they realized that the molecule was a duplex consisting of two anti-parallel strands, with the sugar-phosphate backbone was on the outside, and the bases paired on the inside with hydrogen bonds between A and T, or between C and G, accounting for Chargaff’s rules.

The two strands of a DNA duplex are anti-parallel. Source: Wikimedia Commons (https://commons.wikimedia.org/wiki/File:DNA_chemical_structure.svg), Madprime CC0

The directionality of a strand of DNA (or RNA) can be seen in the sugar-phosphate backbone. One end has a 5′ carbon with a phosphate attached; the other end has a 3′ carbon with a hydroxyl (-OH) group attached. We indicate directionality as 5′ –> 3′. The two strands of DNA are not only complementary to each other (where one strand has an A, the complementary strand has a T and where one strand has a C, the complementary strand has a G, and so forth), but run in opposite directions: 5′–>3′ and 3′ <– 5′.

Structure of double-stranded DNA. Source: Wikimedia Commons (https://commons.wikimedia.org/wiki/File:ADN_animation.gif)

 

Part 3: The Meselson-Stahl experiment revealing the mechanism of DNA replication

Meselson and Stahl demonstrate that DNA replication cannot be conservative or dispersive, must be semi-conservative

The structure of DNA, consisting of two complementary strands, suggests that DNA could replicate by unzipping into two separate strands, and each strand would serve as the template for the synthesis of its complementary partner strand. This is called semi-conservative replication, because each resulting daughter DNA molecule would consist of one original (parental) strand and one newly synthesized strand. Other possible modes would be conservative replication, and dispersive replication.

Three possible modes of DNA replication. Source: Wikimedia Commons (https://commons.wikimedia.org/wiki/File:DNAreplicationModes.png)

Matthew Meselson and Franklin Stahl used a density isotope of nitrogen, 15N, to label DNA and density-gradient ultracentrifugation to analyze 15N density-labeled DNA (heavy DNA) before and after rounds of replication in medium contain normal 14N (light nitrogen). The semi-conservative model predicted that, in this experiment, the daughter DNA molecules after one round of DNA replication would all consist of one 15N-labeled DNA strand and one 14N-labeled DNA strand, and therefore would have intermediate density. That was exactly the result observed.

Meselson and Stahl used a density isotope of nitrogen to label DNA and test alternative hypotheses for how cells replicate their DNA. Source: Wikimedia Commons (https://commons.wikimedia.org/wiki/File:Meselson-stahl_experiment_diagram_en.svg)

Students should work through the results predicted by conservative and dispersive modes of replication, and explain how the observed results disprove these alternative hypotheses.

Here is a video summary of the Meselson-Stahl experiment:

 

Part 4: The enzymes of DNA replication and replication differences at the leading vs lagging strand

DNA replication proceeds bidirectionally from an origin of replication, but the two strands are replicated differently

  • Replication of chromosomal DNA begins at special sites called origins of replication, where the DNA duplex is unzipped. Origins of replication tend to be full of A-T base pairs rather than G-C.  What property of A-T pairing vs G-C pairing could explain this observation?
  • DNA replication proceeds bidirectionally from the origin, with the enzyme helicase at the replication “fork” unzipping and unwinding the DNA template in both directions away from the origin.
  • DNA polymerase requires an RNA primer, synthesized by primase, to begin polymerizing DNA. DNA polymerase has to have an existing 3′-OH to add to, so it cannot start synthesizing a complementary strand from scratch.  But RNA polymerase can start from scratch, so a new DNA strand starts with an RNA primer which is synthesized by primase. DNA polymerase then starts by adding on to the 3′-OH end of the RNA primer.
  • DNA polymerase adds new nucleotides only at the 3′-OH end, so new strands grow 5′ –> 3′.
  • DNA polymerase reads the template strand 3′ –> 5′, so the new DNA duplex is antiparallel.
  • One daughter strand, called the “leading” strand, can be synthesized continuously, in the same direction as the DNA is being unzipped at the fork.
  • The other daughter strand, the “lagging” strand, has to be synthesized in short fragments, called “Okazaki” fragments, that start with an RNA primer and are synthesized going away from the fork.
  • The Okazaki fragments are sealed by an enzyme called DNA ligase.

The DNA replication fork. This fork moves from right to left as DNA is unzipped. a: template, b: leading strand, c: lagging strand, d: replication fork, e: primer, f: Okazaki fragments. Wikipedia image by Masur; CC-BY 2.5

 

As the replication fork extends in one direction above, the leading strand (top strand) can be synthesized continuously because it is proceeding in the 5′ to 3′ direction.  The lagging strand must be synthesized in short fragments as the DNA fork extends.  This is because DNA synthesis must ALWAYS proceed in the 5′ to 3′ direction, but the lagging strand template is running the ‘wrong’ way for synthesis to occur in the direction of the replication fork. Note both strands require RNA primers (red) to begin DNA synthesis.

In the panels below, you can see movement of the replication fork “in action,” and why the leading strand has continuous synthesis while the lagging strand is composed of short fragments based on the direction of DNA synthesis in the 5′ to 3′ direction:

replication-fork-movement

In the image below, you can see that each side of the replication “bubble” or “eye” has a fork, and each fork has a leading and a lagging strand:

Model_of_in_vitro_replication_of_SV40_DNA_by_eukaryotic_enzymes

Source: Adapted from Wikimedia Commons (https://commons.wikimedia.org/wiki/File:Model_of_in_vitro_replication_of_SV40_DNA_by_eukaryotic_enzymes.jpg)

 

Go through this interactive animation; start with “Replication Fork,” then “Fork with Proteins.” Optional: “Concerted Replication,” and “Trombone Model”

http://www.courses.fas.harvard.edu/~biotext/animations/replication1.swf

 

Summary of parts 2 and 4: Crash Course on DNA structure and replication

This video provides an engaging summary of the ideas discussed above for DNA structure and the enzymes involved in DNA replication (see if you can spot the mistake on DNA structure):

 

For more help with these topics, you can view Dr. Choi’s video lectures below:

The experiments proving that DNA is the hereditary material, and the structure of DNA:

The Meselson-Stahl experiment and DNA replication:

Slide set to go with the two lecture videos on this page:

B1510_module4-5_DNA_2011

Put it all together:

forensic_dna_sequencing_2012

Other resources:

Molecular animation of the “trombone” model of DNA replication:

http://www.dnalc.org/resources/3d/04-mechanism-of-replication-advanced.html

 

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7 Responses to DNA

  1. Kyle Lesperance says:

    I believe Hank Green’s mistake was that he circled the 5′ end every time.

    • Jung Choi says:

      Yes, he circles the 5′ end and calls it the 5′ end the first time, then he circles the 5′ carbon on the next nucleotide and calls it the 3′ – about 3:14 into the video.

  2. Abhinav says:

    I have a question:
    Is there a reason for having the two DNA strands anti-parallel? Is it advantageous in any way?

    • Jung Choi says:

      I believe the base-pairing (hydrogen bonds) doesn’t work if the two strands are parallel. The two strands have to be antiparallel for the bases to be in the right orientation to form the hydrogen bonds.

  3. Abhinav says:

    ‘Writing to his son, Michael, Francis Crick tells of a “most important discovery.”’
    http://www.nytimes.com/interactive/2013/02/26/science/crick-letter-on-dna-discovery.html?smid=fb-share&_r=0

  4. noneill3 says:

    “The leading strand must be synthesized in short fragments as the DNA fork extends.”

    Shouldn’t this say “lagging strand”?
    Thanks!

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