1. DNA Replication
Biology 12

2. Cells can contain 6-9 feet of DNA
Cells can contain 6-9 feet of DNA. If all the DNA in your body was put end to end, it would reach to the sun and back over 600 times.
DNA in all humans is 99.9 percent identical. It is about one tenth of one percent that makes us all unique, or about 3 million nucleotides difference.
DNA can store 25 gigabytes of information per inch and is the most efficient storage system known to human. So, humans are better than computers!!
In an average meal, you eat approximately 55,000,000 cells or between 63,000 to 93,000 miles of DNA.
It would take a person typing 60 words per minute, eight hours a day, around 50 years to type the human genome.

3. So What Do We Know?
DNA is composed of units called NUCLEOTIDES, which are composed of three sub-molecules:
1. Pentose Sugar (deoxyribose)
2. Phosphate
3. Nitrogen Base (purine or pyrimidine)

4. Two Kinds of Bases in DNA
Pyrimidines are single ring bases.
Purines are double ring bases. N C O C C N C C N

5. So What Do We Know?
DNA is composed of two complimentary strands of nucleotides joined by hydrogen bonds:
Adenine with Thymine (A-T or T-A)
They join with 2 hydrogen bonds
Cytosine with Guanine (C-G or G-C)
They join with 3 hydrogen bonds
DNA twists into a double helix

6. Chargraff’s Rule: Adenine and Thymine always join together A T
Cytosine and Guanine always join together
C G

7. Bonding 1 10 Adenine Thymine Cytosine Guanine
The bases always pair up in the same way to form base pairs.
Adenine forms a bond with Thymine
Adenine
Thymine
and Cytosine bonds with Guanine
Cytosine
Guanine

8. Bonding 2 11
PO4
adenine
cytosine
PO4
thymine
PO4
guanine
PO4
PO4

10. Replication Copying the genetic material is REPLICATION.
Replication occurs prior to
cell division, because the new, daughter cell will also need a complete copy of cellular DNA.
Cell division
Replication

11. DNA Replication Problem Solving
On the surface…. The replication of DNA is pretty simple. Just unzip, plug in the the spare parts by complementary base pairing, and stitch up the new backbone.
There are a lot of irritating details and problems with this process.
The solutions to these problems involve a list of vital enzymes.

12. DNA Replication Complexities:
DNA is a very stable molecule but its stability depends on its double stranded nature. Single stranded DNA is vulnerable to a number of kinds of damage. So…..to take a huge DNA molecule and seperate its two strands for its entire length....is a very bad idea.
So how can long DNA molecules be replicated without making them single stranded for long periods of time?

13. DNA is very long. VERY long
DNA is very long. VERY long. How can a very long DNA molecule get itself replicated in a relatively short period of time?

14. The primary DNA replication enzyme, DNA Polymerase, is highly specific in a number of ways. One of those specificities is that it can only add new nucleotides to an already existing growing strand of nucleotides. This is a problem because it means that DNA polymerase is not capable of actually starting the process itself. How does replication get started?

15. DNA polymerase is also highly specific in that it can only build new polynucleotide strands in the 5' to 3' direction--new strands must always run from 5' to 3'. But the two strands of DNA are antiparallel to each other--one runs 3' to 5', the other from 5' to 3'. Both sides have to be replicated. How is this puzzle solved in DNA replication? What's the name of the scientist credited with this discovery?

16. DNA is double stranded, and the two strands twist around each other
DNA is double stranded, and the two strands twist around each other. These two strands need to be pulled apart, thus tightening the twist between Origin points. This would lead to accidental breakage of the polynucleotide strands as the twists got compressed into smaller and smaller lengths. How is this problem avoided in DNA replication? What is the name of the enzyme needed to solve this problem (it has several; any of them will do)?

17. Finally, the solutions to the previous problems leave us with an embarrassing problem: single stranded breaks in the polynucleotides of our new DNA molecules. How are these breaks repaired? Again, you'll need to identify the enzyme involved.

18. DNA Replication Overview
Enzyme breaks weak hydrogen bonds
DNA strands open up
Free nucleotides (from our food) fill in the open side (free nucleotides are a significant component of the nucleoplasm in any cell.
Using complimentary
base pairing
End Result:
2 identical DNA strands

19. How Does DNA Replicate? Three Hypotheses: Conservative
Semi-Conservative
Dispersive
2 strands of the parent stay together, daughter gets new two strands
New DNA is made of a random
Mixture of parent and daughter DNA
2 strands of parent separate, daughter gets 1 strand of DNA from parent

20. Meselson-Stahl Discovered the ‘semi-conservative’ model
Parental (old)
DNA molecule
Discovered the ‘semi-conservative’ model
Experimented with bacteria
Replication fork
(site of replication)
Daughter
(new) strand
Daughter
DNA molecule
(double helices)
Figure 10.6

21. Semi-Conservative Replication
Origin of name:
Original parent strands conserved BUT are not still attached together
End Result:
Each new daughter DNA strand is ½ “old” and ½ “new”

22. Three Steps In DNA Replication
1. Initiation – Replication begins at a location on the double helix known as “oriC” to which a certain initiator proteins bind and trigger unwinding. Enzymes known as helicases unwind the double helix by breaking the hydrogen bonds between complimentary base pairs, while other proteins keep the single strands from rejoining. The topoisomerase proteins surround the unzipping strand and relax the twisting that migh damage the unwinding DNA.

23. 2. Elongation
With the primer as the starting point for the leading strand, a new DNA strand grows one base at a time. The old (existing) strand is the template for the new strand. The enzyme DNA polymerase controls elongation, which can only occur in the leading direction.
The lagging strand unwinds in small sections that DNA polymerase replicates in the leading direction. The resulting “Okazaki fragments” can contain between 100 to 200 bases. The fragments terminate in an RNA primer that is later removed so that enzymes stitch the back together into one long strand.

24. 3. Termination
After the elongation is completed, two new double helices have replaced the original one.
During termination the last primer must be removed from the end of the lagging strand.
Enzymes proofread the new double helix and remove mispaired bases.

25. Step #1 Separating DNA Strands
Gyrase
Relieves tension by unwinding
Helicase
Breaks hydrogen bonds
Unzips the DNA
Terminates at fork
- Enzyme breaks the weak hydrogen bonds
Splitting the parent DNA strand
Leaving two separated strands

26. Separating DNA Strands
SSBs (Single-stranded binding proteins)
Bind to the exposed DNA
Keep from “annealing”
Reattaching with complimentary base pair

27. The two sides of the molecule are
separated for a short distance.
Since DNA is most stable (and
least vulnerable to damage) in its
double stranded configuration,
as little of it as possible will be
single stranded at once.

28. DNA Replication Priming:
1. RNA primers: before new DNA strands can form, there must be small pre-existing primers (RNA) present to start the addition of new nucleotides (DNA Polymerase).
2. Primase: enzyme that polymerizes (synthesizes) the RNA Primer.

29. DNA polymerase performs only one job, following the complementary
base pairing rule. It adds the new free nucleotides in the new strand
of a replicating DNA molecule.
This also means that DNA polymerase cannot actually start the process
of replication.
An enzyme called primase (an RNA polymerase) actually begins the
replication process. It builds a short piece of RNA called a primer. This
primer is later removed by RNAse H and replaced by DNA
nucleotides.

30. Step #2 Building Complimentary Strands
Replication proceeds along
Both sides of the replication
fork.
Free nucleotides with the assistance of a protein called DNA polymerase, attach to original parent DNA strand which serves as a template.
Nucleotides in the new strand are selected using complimentary base pairing
A with T and C with G

31. DNA polymerase is only capable of building a new strand from the 5’ end to the 3’ end. This is a problem because the two sides of the DNA are antiparallel.
One side of the new strand (called the leading strand) can be directly and continuously constructed from its 5’ end to its 3’ end.

32. DNA Replication Synthesis of the new DNA Strands:
1. DNA Polymerase: with a RNA primer in place, DNA Polymerase (enzyme) catalyze the synthesis of a new DNA strand in the 5’ to 3’ direction.
RNA
Primer
DNA Polymerase
Nucleotide 5’ 3’

33. DNA Replication
2. Leading Strand: synthesized as a single polymer in the 5’ to 3’
direction.
RNA
Primer
DNA Polymerase
Nucleotides 3’ 5’

34. Figure 5 Single-stranded binding proteins Parent strand
Replication fork
III

35. The other strand (called the lagging strand) must be constructed in short segments, built backwards.
These short strands of new DNA are called Okasaki fragments after the man who discovered them.
The Okasaki fragments will eventually be connected by an enzyme named DNA ligase. DNA ligase specializes in healing single stranded nicks in DNA. It simply seals the bond between one nucleotide and the neighboring nucleotide.

36. DNA Replication
3. Lagging Strand: also synthesized in the 5’ to 3’ direction, but discontinuously against overall direction of replication.
RNA Primer
Leading Strand
DNA Polymerase 5’ 3’
Lagging Strand 5’ 3’

37. DNA Replication
4. Okazaki Fragments: series of short segments on the lagging strand.
Lagging Strand
RNA
Primer
DNA
Polymerase 3’ 5’
Okazaki Fragment

38. DNA Replication
5. DNA ligase: a linking enzyme that catalyzes the formation of a covalent bond from the 3’ to 5’ end of joining stands.
Example: joining two Okazaki fragments together.
Lagging Strand
Okazaki Fragment 2
DNA ligase
Okazaki Fragment 1 5’ 3’

39. Joins Okazaki fragments together
DNA ligase
Joins Okazaki fragments together
Completes backbone
Lagging strand only!

40. Building Complimentary Strands
Leading strand
Built continuously
From parent 3’ end toward replication fork

41. Building Complimentary Strands
Lagging strand
Built in short Okazaki fragments between RNA primers
Discontinuous
From parent 3’ end away from
replication fork

42. Single-stranded binding proteins
Okazaki fragments
Parent strand
Replication fork
III

43. Step 1
Step 2
Step 3

44. DNA is extremely long. It would take a very long time to replicate the whole molecule from end to end using only a single replication fork.
Each of these long molecules have many sites called Origins. This forms a configuration called a replication bubble. Each replication bubble actually has two forks, one on each end of the bubble and travelling in opposite directions.

45. Two daughter DNA molecules
Fork vs. Bubble
Origin of
replication
Origin of
replication
Replication Bubble:
Begins at multiple sites
Produces multiple forks
Proceeds in both directions
Result:
Faster replication
Origin of
replication
Parental strand
Daughter strand
Bubble
Two daughter DNA molecules
Figure 10.8

47. DNA cannot be double stranded and not twist. Unless
there is a way to relax the twists between replication
bubbles, the helical twists would compress putting
increasing stress on the molecule causing random breaking
and damage. This problem is solved by the creation of single
stranded breaks (swivels) between origin sites.
These nicks are made by an enzyme called
DNA topoisomerase (previously called “unwindase”,
then “swivelase”.
After the replication bubbles meet, ‘the single strands are
healed by DNA Ligase.

48. Proofreading DNA polymerase I and III Check for proper base pairing
If mistake found:
Removes
Replace with proper nucleotide

49. End Result Results in two identical daughter DNA strands
½ new and ½ old

50. Leading vs. Lagging Leading Strand Lagging Strand
Toward replication fork
Direction complimentary strand is built
Away from replication fork
Yes
Built continuously? No
(Okazaki fragments)
Only once
Number of times RNA primers are needed
Multiple times
(as replication fork moves)

51. Leading vs. Lagging From daughter 5’ to 3’ end Lagging ONLY Both:
DNA ligase completes backbone bonds
Both:
Built from parent 3’ to 5’ end
From daughter 5’ to 3’ end
Use DNA polymerase III & I

52. Leading vs. Lagging Why do we need leading and lagging strands?
DNA polymerase III can only build from parent 3’ end
DNA runs antiparallel
When “unzipping”:
Leading: 3’ end IS available
Lagging: 3’ end IS NOT available

53. Human Genome 3 BILLION base pairs
In each cell = 46 DNA strands
Mistakes can occur with mismatched pairs
Called mutations

54. DNA Repair If no repair In sex cells  inherited diseases
Example Tay-Sachs disease - the body lacks hexosaminidase A, a protein that helps break down a chemical found in nerve tissue
caused by a defective gene on chromosome 15
In somatic cells  cancer

55. Replication Mistakes
Change in the nucleotide base sequence of a genome; rare.
Almost always deleterious (bad). A deleterious mutation has a negative effect on the phenotype, and thus decreases the fitness of the organism. (A harmful mutation)
Rarely lead to a protein having a novel property that improves ability of organism and its descendents to survive and reproduce.
Mutations

56. Effect of Mutation Sickle cell anemia is a disease passed
down through families
in which red blood
cells form an abnormal
sickle or crescent
shape. Red blood cells
carry oxygen to the
body and are normally
shaped like a disc.
Causes bone pain.