1. DNA History, Structure, and Replication

2. Where is genetic information stored?*
QUESTION:
Where is genetic information stored?*
a) in the ribosomes of cells
b) within the proteins of cells
c) within the DNA of cells
d) within the membrane of cells
The answer is here somewhere!
*But scientists did not always know this! DNA is a very small molecule and had to be discovered. Let’s look at the history, structure, and replication of DNA in today’s discussion.

3. Discovering DNA -
Once Mendel understood that “factors” could be passed to offspring, scientists began to wonder what these factors were. They were sure of a few things…
The factors needed to be able to store information
The factors needed to be replicable
And the factors needed to sometimes undergo changes (mutations)

4. 1869 – Discovering Nucleic Acids
Swiss Physician, Johannes Friedrich Miescher isolated a new biomolecule he called “nuclein” from the nuclei of white blood cells. This later became called nucleic acids, but to be honest – we had no idea what it did. We just knew it was there.
Miescher used pus and bloodstained bandages from a hospital to study “nuclein”

5. Chemistry of NUCLEOTIDES (Nucleic Acid Monomers)
Analysis of the nucleic acid showed that it contained a sugar, phosphate and one of four nitrogen bases
Adenine
Guanine
Cytosine
Thymine
- Now we know what’s in it, but what does it look like? What does it do?

6. **THE BIG QUESTIONS**
Were these new nucleic acids the secret to Mendel’s factors? Or were the ever-present proteins the secret? Where is the cell’s genetic code?
a) Proteins contain 20 amino acids that can be organized in countless ways to determine traits
b) Nucleic acids only contained 4 different nucleotides

7. Thanks to their 20 amino acids, scientists were leaning toward proteins.
As a information storage option, amino acids would have a lot of options, since you could combine these in different ways
The 4 bases of the NAs didn’t seem to have as much versatility.
It’s like have an alphabet of 20 letters versus and alphabet of 4.

8. The Experiment that finally gave us an answer…
Frederick Griffith was attempting to find a vaccine against pneumococcus bacteria that caused pneumonia. In this experiment, he accidentally determined the true source of the genetic code by discovering that one type of bacteria could actually turn into another. Let’s take a look at his work…

9. An overview of the transformation experiment.
Can you summarize this in your own words?

10. DNA was determined to be the genetic code.
DNA from the dead S strain bacteria was taken in by the live R strain, causing them to transform into S strains.
Denaturing the proteins or using enzymes that stopped S proteins did not stop the transformation
Using enzymes that denatured the nucleic acid did stop the transformation
We knew the DNA contained the instructions, but we still didn’t understand how… All that information transferred using only four “letters”?! How is that possible?!

11. 2) What was Miescher’s contribution to genetic studies?
Quick Recap:
1) What caused scientists to believe that proteins contained the genetic code?
2) What was Miescher’s contribution to genetic studies?
3) How were the nonvirulent R strain bacteria transformed into a virulent strain?
4) Griffith’s experiment resulted in which conclusion?

12. Alfred Hershey and Martha Chase Experiments

13. The bacteriophage – a virus used for studying DNA
Reminder: Viruses infect by injecting their DNA into a cell and taking control of the host. Two types– Lytic viruses immediately use the host to replicate their own DNA, make more viruses, and then lyse the cell to release offspring. Lysogenic viruses actually insert their DNA into the genome of the host. They go lytic eventually, but for a while, they “hide” in the host DNA and become a part of the host organism. The bacteriophage is a lysogenic virus for bacteria. How could we use them?

14. Bacteriophages – viruses that infect bacteria
Using the bacteriophage to prove DNA as the genetic code
Bacteriophages have a protein capsid surrounding a piece of DNA
Experiments used radioactive sulfur to tag proteins and radioactive phosphorous to tag the DNA.
The goal was to see which substance (protein or DNA) moved into the infected cell

15. Conclusion:  The radioactive tag on the DNA went into the bacteria, not the tagged proteins

16. But we still had NO clue what it looked like!
Imagine you had all of the pieces to a puzzle but you didn’t know how they fit together. Scientists had the pieces of DNA. Fame and fortune would go to the one who solved this puzzle...

17. The Race to Establish the Structure of DNA
THE PLAYERS
THE PIECES
adenine
guanine
cytosine
thymine
deoxyribose
phosphate

18. Examine the data below. Do you notice a pattern?

19. Chargaff’s Rule So did Erwin Chargaff...
all species had similar ratios of A, T, G, C
Chargaff’s Rule
Amount of A, T, G, C varies by species, but #A = #T and #G = #C (#Purines = #Pyrimidines)

20. Purines (A&G) have two rings on the nitrogen base

21. Pyrimidines (C&T) have one ring on the nitrogen base

22. Could it be that these pieces of the puzzle fit together…..
But what about deoxyribose and phosphates, where do those pieces fit? And what is the shape of DNA? C G T A G C G C A T

23. ROSALIND FRANKLIN & WILKENS
Took pictures of DNA structure with X-RAY DIFFRACTION

24. These images provided clues to the shape of the DNA molecule.

25. Competition in science was a major theme during this period of time
Competition in science was a major theme during this period of time. ( ish)
Scientists often wanted to get sole credit for a discovery, and were reluctant to share results with others. None of the work by Chargaff, Franklin, and Wilkins were shared, they existed as isolated facts.
For consideration…..
1) Do you think scientists today are more likely to collaborate?
2) How has the internet changed science?

26. Enter……...WATSON & CRICK

27. They were criticized for their methods which included
- hanging out in pubs and talking about stuff
- playing cricket
- stealing data from other scientists (Franklin/Wilkins)
- But they used Chargaff’s rules and F/W’s picture to mathematically determine the double helix shape of DNA. They won the Nobel Prize for “solving the puzzle” and mostly- they get the credit. Fair?

28. DNA: DOUBLE HELIX (Twisted Ladder)
Steps of ladder are bases (A, T, G, C)
Sides of ladder are sugar & phosphate
Sugar and phosphate are covalently bonded along the sides (strong!) while the steps are hydrogen bonded (weak.)

29. 5' and 3' ends 5' 4' 1' 3' 2' 2' 3' 1' 4'
Each side is antiparallel (runs in the opposite direction). The numbers used to represent each side refer to the carbons attached to the deoxyribose and covalently bonded to the phosphate. If it’s connected at the 3rd Carbon, it’s the 3’ end. Vice versa for 5’. 5'

30. Each Side is ANTIPARALLEL
5’ and 3’ ENDS
Each Side is ANTIPARALLEL

31. Nucleotide = 1 base Deoxyribose (sugar) 1 phosphate
DNA is a polymer made
from nucleotide
monomers

34. What’s wrong with this drawing?

35. DNA REPLICATION
-the process by which DNA makes a copy of itself during S phase of interphase prior to ANY cell division
-replication is semi-conservative, because one half of the original strand is always saved, or "conserved” in the new strands
Looking at the pic, explain replication in your words.

36. DNA Replication Steps
1. Protein enzyme(s) DNA helicase unzips the hydrogen bonds and creates replication forks at several places along the molecule. DNA binding proteins hold the separated strands apart to prevent reannealing (reattaching).
2. Protein enzyme Primase creates a small section of nucleotides to which protein enzymes DNA polymerases can fully attach and run down the strand adding free nucleotides (following base pair rules) to the exposed strand, covalently binding the sugars and phosphates on the new side, and proofreading/correcting mistakes along the way.
***DNA polymerase can only travel down the strand from the 3' to the 5' end. Careful: it only “reads” the OLD strand from 3-5, meaning it “builds” the NEW strand from What issue does this create?***

37. 3. Since one side of the DNA runs in the 3’ to 5’ direction, it is copied continuously and called the leading strand. The other side runs in the 5' to 3' direction and is called the lagging strand. Since the DNA polymerase can only READ from 3’ to 5’ and BUILD from 5’ to 3’, this lagging strand must be done in chunks called OKAZAKI FRAGMENTS. Primase places a primer periodically, and DNA Polymerase “jumps” from primer to primer working in reverse. These fragments are eventually connected by another protein enzyme called DNA LIGASE 4. Using multiple replication forks (sometimes called replication bubbles) all down the strand, these enzymes are able to copy all of the DNA relatively quickly.

38. DNA Replication

39. End of the Line Issues
Presence of RNA primer on the 5’ ends of daughter DNA leading strand leaves a gap of uncopied DNA
Repeated rounds of replication produce shorter and shorter DNA molecules (possible cause of aging?)
Telomeres are enzymes found only in gamete producing cells that
protect genes from being eroded through multiple rounds of DNA replication for offspring

40. Telomeres
Ends of eukaryotic chromosomes, the telomeres, have special nucleotide sequences
Humans - this sequence is typically TTAGGG, repeated ,000 times (not an actual used gene)
Telomerase adds a short molecule of RNA as a template to extend the 3’ end
Now there is room for primase & DNA pol to extend to the end

41. Summary
Explain how the cell overcomes each of the following issues in DNA replication
DNA strands must be unwound during replication
A new DNA strand can only elongate in the 5’  3’ direction
DNA polymerase cannot initiate synthesis and can only add nucleotides to end of an existing chain
Presence of RNA primer on the 5’ ends of daughter DNA leading strand leaves a gap of uncopied DNA

42. Which side is leading. Which side is lagging
Which side is leading? Which side is lagging? Which side will be made continuously? Which side will be made in Okasaki fragments?

43. Pg 235

44. Figure 13Ac