1. By the end of this lesson…
You should be able to describe the structure of DNA in detail.
You should be able to narrate the replication of a DNA molecule including all enzymes used therein.

2. DNA Structure Review
DNA is a nucleic acid, a long string of nucleotides.
DNA takes the shape of a double-helix.
There are four kinds of nucleotides:
Adenine
Cytosine
Guanine
Thymine
Fun fact! Guanine was discovered in animal waste…hence the name.

3. Nucleotide Structure Review
Each nucleotide has a:
Sugar molecule with 5-carbons (pentose)
Deoxyribose in DNA
Ribose in RNA
Phosphate group
Phosphorous-based molecule
Nitrogenous base (makes the nucleotide unique)
Adenine
Thymine
Cytosine
Guanine

4. Nucleotide Structure Review
SKETCH ME! I’m useful!
SKETCH ME! I’m useful!
Nucleotide Structure Review
SKETCH ME! I’m useful!
SKETCH ME! I’m useful!
Guanine
Cytosine
Thymine
Adenine
SKETCH ME! I’m useful!
SKETCH ME! I’m useful!

5. Nucleotide Structure Review
More “scientific”

6. Nucleotides and Nucleosides
Just so you know, you’ll occasionally hear of a nucleoside.
The only difference between a nucleoside and a nucleotide is that a nucleoside is just a sugar and nitrogenous base – no phosphate group.

7. DNA Structure Review
Surrounding the base pairs and forming the sides of the “ladder” is a sugar-phosphate backbone.
The backbone is made of a sugar (deoxyribose) and a phosphate group, alternating and in reverse order from the other strand.
Backbone is linked by phosphodiester bonds.
The end of DNA with the phosphate on top is the 5’ (“five prime”) end.
The other end of the backbone is the 3’ (“three prime”) end.

8. 3’ and 5’? Huh?
3’ and 5’ get their names from the pentose sugar’s carbon atoms.
Each carbon in pentose is numbered and has a specific job in the formation of DNA.
Carbon 1 = base attachment
Carbon 2 = oxygen (ribose) or not (deoxyribose)?
Carbon 3 = another nucleotide attachment
Carbon 4 = completes ring
Carbon 5 = phosphate attachment
This is important.

9. One more time, because “important.”
 Oxygen, not a zero.

10. DNA Unwound ---H--- ---H--- ---H--- P Adenine Thymine P Guanine
Deoxy-ribose P
Adenine
Thymine
Deoxy-ribose P
Guanine
Adenine 5’ 3’
---H---
DNA Unwound
PD Bond
Deoxy-ribose P
Cytosine
PD Bond
---H---
PD Bond P
PD Bond
Deoxy-ribose
---H---
Thymine 5’ 3’

11. Antiparallel Strands
As seen in the image to the right, the two strands of DNA run antiparallel to one another.
One is “upside down.”
At the 5’ end of each DNA strand there is a phosphate group.
At the 3’ end of each DNA strand there is a hydroxyl (-OH) group.

12. Bonding in DNA
Phosphodiester Bond
Hydrogen Bond
The two complementary strands of DNA are linked by hydrogen bonds.
Base to base.
Each nucleotide in a sugar-phosphate backbone is linked by a phosphodiester bond.
Phosphate group to 3’ C.

13. Linking Nucleotides
Phosphodiester bonds, linking nucleotides, are formed…how?
By dehydration synthesis, of course!
More on this later.

14. Purines and Pyrimidines
Adenine and guanine are purines and have a double-ring structure.
Cytosine and thymine are pyrimidines and have a single-ring structure.
A purine always bonds to a pyrimidine.
This ensures that the width of the double helix is constant.
How can we remember this one?

15. DNA Replication
There comes a time in (almost) every DNA molecule’s life when it needs to be replicated (copied).
That time would be S phase.
Here’s the general process:
Unwind the double-helix.
Break the hydrogen bonds (“unzip” the DNA).
Use enzymes to replace base pairs on each side.

16. DNA Polymerase makes new DNA DNA Helicase breaks H-bonds
Thymine
Deoxy-ribose P
Guanine
Adenine
Deoxy-ribose P
Thymine
Cytosine
Adenine P
Deoxy-ribose
Thymine
Deoxy-ribose
Adenine H
---H--- H
Replication
Deoxy-ribose P
Cytosine
DNA Polymerase makes new DNA
Strands move apart
DNA Helicase breaks H-bonds
Deoxy-ribose
Guanine P H
---H--- H
Deoxy-ribose P
Thymine
Deoxy-ribose
Adenine P H
---H--- H P

17. Looks like this… [IMPORTANT]
The “old” strand is sometimes known as the template strand because it’s a model for the new one.
Note that even though there are two strands forming down here, each is only “half” new.

18. Looks like this… [IMPORTANT]

19. Replication Enzymes Helicase DNA Polymerase III (abbreviated pol III)
DNA Polymerase I (abbreviated pol I)
Ligase
Primase
Technically, more than a dozen enzymes participate in replication.
Many are smaller enzymes complexed into larger ones.

20. ATP, GTP, CTP, and TTP
DNA polymerization uses ATP, along with its close relatives GTP, CTP, and TTP – each corresponding to a different letter.
In other words, the energy is packed with the raw materials.
DNA bases arrive as nucleosides (nucleotides without the single phosphate), and in fact have three phosphate groups attached.
We call them nucleoside triphosphates:
Adenosine Triphosphate
Guanosine Triphosphate
Thymidine Triphosphate
Cytidine Triphosphate

21. Formation of Phosphodiester Bonds
Deoxy-ribose P
Thymine
Formation of Phosphodiester Bonds P
Deoxy-ribose P
Adenine
 Dehydration Synthesis P
Deoxy-ribose P
Guanine
 Dehydration Synthesis

22. DNA Replication Process Summary Slide [enzymes underlined]
DNA helicase unwinds the double helix by breaking hydrogen bonds between nitrogenous bases.
Single-strand binding proteins prevent re-coiling.
Topoisomerase relieves physical strain in the coiled part of the strand.
Primase lays down an RNA nucleotide primer.
Pol III adds DNA nucleotide bases from 5’ to 3’.
Pol I replaces RNA primers with DNA nucleotides.
Ligase joins disconnected fragments of DNA.

23. Close-Up On Next Slides
More on Replication
Close-Up On Next Slides 3’ 5’
Replication Fork
Replication Fork 5’ 3’
Note: The following slides concerning replication will feature close-ups of different regions of the above molecule as it is replicated, unless otherwise noted. That’s important to know.

24. DNA Replication: Leading Strand
For the leading strand, primase lays down an RNA primer (5’ to 3’). 5’
Pol III adds DNA nucleotides (5’ to 3’). 3’ 3’ 5’
Pol III
Primase 5’ 3’

25. DNA Replication: Leading Strand5’
As the replication fork moves toward the 3’ end, Pol III adds more nucleotides continuously. 3’ 3’ 5’
Pol III 5’ 3’

26. DNA Replication: Lagging Strand
For the lagging strand, primase lays down an RNA primer (5’ to 3’). 5’
Pol III 3’
Pol III adds DNA nucleotides (5’ to 3’).
Primase 3’ 5’ 5’ 3’

27. DNA Replication: Lagging Strand5’
As the replication fork moves toward the 5’ end of the daughter strand, a new primer is needed. 3’
We have now formed two Okazaki fragments.
Pol III
Primase 3’ 5’ 5’ 3’

28. DNA Replication: Leading and Lagging Strands
Leading Strand 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’
Lagging Strand
Leading Strand

29. Step 4: Pol I Replaces Primers5’
Pol I moves in and changes all the RNA primers to segments of DNA. 3’
Pol I
Pol I 3’ 5’ 5’
Pol I 3’

30. Step 5: Ligase Polymerization
Ligase joins Okazaki fragments to seal any gaps in the DNA. 3’
Ligase 3’ 5’ 5’
Note:
These gaps are actually just missing phosphodiester bonds, not missing nucleotides. Ligase does not add any nucleotides. 3’

31. So once again, that’s…
DNA helicase unwinds the double helix by breaking hydrogen bonds between nitrogenous bases.
Single-strand binding proteins prevent re-coiling.
Topoisomerase relieves physical strain in the coiled part of the strand.
Primase lays down an RNA nucleotide primer.
Pol III adds DNA nucleotide bases from 5’ to 3’.
Pol I replaces RNA primers with DNA nucleotides.
Ligase joins disconnected fragments of DNA.

32. Or, in one image…

33. Step 1: Unwind the DNA
For protection, the DNA molecule is highly coiled.
A consequence, however, is that it also can’t be copied – enzymes cannot access it.
DNA helicase uncoils the helix and creates two replication forks (uncoiling spots).
Sometimes called a “replication bubble.”
There are many replication bubbles all throughout a replicating DNA molecule, all at the same time.

34. Step 1: Unwind the DNA
Remember that we’re dealing with unthinking molecules, though. How does the DNA molecule not just re-coil?
Single-strand binding proteins attach themselves to the nucleotides to prevent them from coming back together.
AND ALL FOLLOWING

35. Step 1: Unwind the DNA
Of course, untwisting one section of the DNA will add strain to the still-coiled section.
Topoisomerase prevents damage to the “upstream” part of the strand.

36. Step 2: Add RNA Primers  3’ 5’
DNA polymerase (III and I) has a major limitation:
It needs a 3’ carbon to serve as a foundation for the placement of the 5’ end.
It’s really that little hydroxyl group that it needs to “plug into.”
So even though the parent DNA molecule is ready to go, there’s no way for pol III to start adding nucleotides.
Luckily, there’s another enzyme out there that can get things started: primase.   5’ 3’

37. Step 2: Add RNA Primers
Primase attaches to the DNA molecule and adds a short stretch of RNA to the template parent strand (5-10 nucleotides).
These primers provide the 3’ carbon for pol III.

38. Step 3: Pol III Polymerization
Pol III, which is made of a bunch of subunits, starts at the primer and adds DNA nucleotides, moving from 5’ to 3’.

39. Step 3: Pol III Polymerization
Wait…what about the 3’ to 5’ direction?
As in, how does pol III polymerize the complementary strand?
Pol III does so in short stretches.
Let’s look at this problem with a conceptual diagram.

40. Why Only 5’ to 3’? 5’ P Thymine P P  Dehydration Synthesis P P 5’
Deoxy-ribose P
Thymine 5’
Why Only 5’ to 3’? P P
Deoxy-ribose P
Guanine
Deoxy-ribose P
Adenine
 Dehydration Synthesis 5’ P
 No Dehydration Synthesis!
Deoxy-ribose P
Guanine
 Dehydration Synthesis
Deoxy-ribose P
Cytosine 3’ 3’

41. Linking Nucleotides
Remember this?

42. 5’ to 3’ Confusion Point Look at the DNA molecule below.
To some of you, DNA polymerase may appear to be running backward.
If so, it’s because you’re looking at the template strand…
…not the daughter strand.
Key: Always look at the daughter strand.
Pol III reads 3’ to 5’ but
writes 5’ to 3’.

43. Leading vs. Lagging Notes
Reiji Okazaki
Leading vs. Lagging Notes
The leading strand is the one polymerized continuously from 5’ to 3’ in the direction of helicase’s movement.
The lagging strand is the one polymerized in sections.
In the opposite direction of helicase’s movement.
The sections are called Okazaki fragments.
The lagging strand is still polymerized at the same speed but takes slightly longer to finish (more on that soon).
It’s also still polymerized 5’ to 3’, just not continuously.