1. DNA and the Gene: Synthesis and Repair15
DNA and the Gene: Synthesis and Repair
Lecture Presentation by Cindy S. Malone, PhD, California State University Northridge

2. Please highlight all the terms in blue.
When you are done with the notes:
Read and study chapter 15 in the textbook.
Read and review your notes.
Come prepared for discussion and activities in class.

3. DNA’s Primary Structure
The primary structure of DNA has two major components:
A backbone made up of the sugar and phosphate groups of deoxyribonucleotides.
A series of nitrogen-containing bases that project from the backbone.
DNA has directionality:
One end has an exposed hydroxyl group on the 3 carbon of deoxyribose and the other end has an exposed phosphate group on a 5 carbon.
The molecule thus has a 3 end and a 5 end.

4. (a) Structure of deoxyribonucleotide
Figure 15.3
(a) Structure of deoxyribonucleotide
Phosphate group attached to 5 carbon of the sugar
Could be adenine (A), thymine (T), guanine (G), cytosine (C)
Hydroxyl (OH) group on 3 carbon of the sugar
(b) Primary structure of DNA
5 end of strand
Nitrogen- containing bases project from the backbone
Sugar-phosphate backbone of DNA strand
Figure 15.3 DNA’s Primary Structure.
Phosphodiester bond links deoxyribonucleotides
3 end of strand 4

5. DNA’s Secondary Structure
Watson and Crick proposed:
Two DNA strands line up in the opposite direction to each other.
This is called antiparallel fashion.
The antiparallel strands twist to form a double helix.
The secondary structure is stabilized by complementary base pairing.
Adenine (A) hydrogen bonds with thymine (T)
Guanine (G) hydrogen bonds with cytosine (C)

6. (a) Complementary base pairing (b) The double helix
Figure 15.4
(a) Complementary base pairing
(b) The double helix
Sugar- phosphate “backbone” of DNA
Complementary base pairs held together by hydrogen bonding
Figure 15.4 DNA’s Secondary Structure: The Double Helix.
Antiparallel strands (their 5  3 polarities run in opposite directions) 6

7. DNA Strands Are Templates for DNA Synthesis
Watson and Crick suggested:
The existing strands of DNA served as a template (pattern) for the production of new strands.
Bases were added to the new strands.
According to complementary base pairing.

8. How Do the Old and New DNA Strands Interact?
In semiconservative replication:
The parental DNA strands separate
Each is used as a template for the synthesis of a new strand
Daughter molecules each consist of one old and one new strand

9. Enzymes for DNA Synthesis
DNA polymerase is the enzyme that catalyzes DNA synthesis.
The discovery of DNA polymerase cleared the way for understanding DNA replication reactions.

10. Characteristics of DNA Polymerases
A critical characteristic of DNA polymerases is that they can work only in one direction.
DNA polymerases can add deoxyribonucleotides to only the 3′ end of a growing DNA chain.
DNA synthesis always proceeds in the 5′  3′ direction.
DNA polymerization is exergonic because the monomers that act as substrates in the reaction are deoxyribonucleoside triphosphates (dNTPs).
They have high potential energy because of their three closely packed phosphate groups.

11. Figure 15.6 The DNA Synthesis Reaction.
Parental strand
Parental strand
3 end
3 end
Daughter strand
Daughter strand
5 end
5 end
Phosphodiester bond
3 end
Figure 15.6 The DNA Synthesis Reaction.
5 end
5 end
Synthesis reaction
3 end
dNTP 11

12. How Does Replication Get Started?
A replication bubble forms in a chromosome that:
Is actively being replicated
Grows as DNA replication proceeds
Because synthesis is bidirectional
In bacterial chromosomes, the replication process begins at a single location.
This is the origin of replication
Eukaryotes also have bidirectional replication but they have multiple origins of replication and they have multiple replication bubbles.
A replication fork is the Y-shaped region where the DNA is split into two separate strands for copying.

13. Replication proceeds in both directions
Figure 15.7
(a) DNA being replicated
(b) Bacterial chromosomes have a single origin of replication.
Old DNA
New DNA
Replication proceeds in both directions
Origin of replication
0.25 m
(c) Eukaryotic chromosomes have multiple origins of replication.
Replication fork
Figure 15.7 DNA Synthesis Proceeds in Two Directions from an Origin of Replication.
Replication bubble
Old DNA
New DNA
Replication proceeds in both directions from each starting point 13

14. How Is the Helix Opened and Stabilized?
Several proteins are responsible for opening and stabilizing the double helix.
Enzyme helicase catalyzes the breaking of hydrogen bonds between the two DNA strands to separate them.
Single-strand DNA-binding proteins (SSBPs) attach to the separated strands to prevent them from closing.
Unwinding the DNA helix creates tension farther down the helix.
Enzyme topoisomerase cuts and rejoins the DNA downstream of the replication fork relieving this tension in the helix.

15. How Is the Leading Strand Synthesized?
DNA polymerase requires a primer:
A few nucleotides are bonded to the template strand
This provides a free 3 hydroxyl (OH) group that can combine with an incoming dNTP to form a phosphodiester bond.
Primase:
A type of RNA polymerase that synthesizes a short RNA segment that serves as a primer.
DNA polymerase III then adds bases to the 3 end of the primer.

16. How Is the Leading Strand Synthesized?
The product is called the leading strand, or continuous strand.
It leads into the replication fork
It is synthesized continuously in the 5′  3′ direction
Primase synthesizes RNA primer
Topoisomerase relieves twisting forces
1. DNA is opened, unwound, and primed.
Helicase opens double helix
Single-strand DNA-binding proteins (SSBPs) stabilize single strands
Sliding clamp holds DNA polymerase in place
DNA polymerase works in 5  3 direction, synthesizing leading strand
RNA primer
2. Synthesis of leading strand begins.
Leading strand

17. The Lagging Strand The other DNA strand is called the lagging strand:
It is synthesized discontinuously
In the direction away from the replication fork
It occurs because DNA synthesis must proceed in the 5  3 direction

18. How Is the Lagging Strand Synthesized?
Synthesis of the lagging strand starts when:
Primase synthesizes a short stretch of RNA and acts as a primer.
DNA polymerase III then adds bases to the 3 end of the primer.
DNA polymerase moves away from the replication fork.
Helicase continues to open the replication fork and expose single-stranded DNA on the lagging strand.

19. Time 1 Time 2 Leading strand synthesized 5  3
Figure 15.9
Leading strand synthesized 5  3
Time 1
Lagging strand synthesized 5  3
Figure 15.9 The Lagging Strand Is Synthesized in a Direction Moving Away from the Replication Fork.
Time 2
Regions of single stranded DNA 19

20. The Discovery of Okazaki Fragments
The lagging strand is synthesized as short discontinuous fragments called Okazaki fragments.
DNA polymerase I removes the RNA primer at the beginning of each Okazaki fragment and fills in the gap.
The enzyme DNA ligase joins the Okazaki fragments to form a continuous DNA strand.
Because Okazaki fragments are synthesized independently and joined together later the lagging strand is also called the discontinuous strand.

21. 2. First fragment synthesized.
Figure 15.10
The leading strands are faded out to help you focus on synthesis of the lagging strand
RNA primer
1. Primer added.
Topoisomerase
SSBPs
Helicase
Primase
Okazaki fragment
2. First fragment synthesized.
Sliding clamp
DNA polymerase III
2nd Okazaki fragment
3. Second fragment synthesized.
1st Okazaki fragment
Figure Lagging-Strand Synthesis.
4. Primer replaced.
DNA polymerase I
DNA ligase
5. Gap closed. 21

22. DNA Synthesis Enzymes Are Well-Organized
The replisome:
The enzymes responsible for DNA synthesis around the replication fork
Joined into one large, multi-enzyme machine

23. Replicating the Ends of Linear Chromosomes
Telomere
Region at the end of a linear chromosome does not contain genes but rather consists of short, repeating stretches of bases.
Replication of telomeres can be problematic:
Leading-strand synthesis results in a normal copy of the DNA molecule.
The telomere on the lagging strand shortens during DNA replication.

24. Replicating the Ends of Linear Chromosomes
Telomeres are the regions at the ends of linear chromosomes.
Replication fork reaches the end of a linear chromosome.
There is no way to replace the RNA primer from the lagging strand with DNA because there is no available primer for DNA synthesis.
The primer is removed leaving a section of single-stranded DNA (lagging strand) at one end of each new chromosome.
Remaining single-stranded DNA is eventually degraded and this results in shortening of the chromosome.

25. 1. DNA unwinding completed.
Figure 15.12
DNA polymerase
End of chromosome
Leading strand
Sliding clamp
1. DNA unwinding completed.
Lagging strand
Helicase
2. Leading strand completed.
RNA primer
Primase
Figure Chromosomes Shorten during Normal DNA Replication.
3. Lagging strand completed.
DNA polymerase
Last Okazaki fragment
4. Lagging strand too short.
No primer for DNA polymerase; unreplicated end will degrade, shortening chromosome
Unreplicated end 25

26. Replicating the Ends of Linear Chromosomes
The enzyme telomerase adds more repeating bases to the end of the lagging strand catalyzing the synthesis of DNA from an RNA template carried with it.
Primase then makes an RNA primer.
DNA polymerase uses primer to synthesize the lagging strand.
Ligase connects the new sequence and prevents the lagging strand from getting shorter with each replication.

27. Missing DNA on lagging strand
Figure 15.13
Missing DNA on lagging strand
1. End is unreplicated.
Telomerase with its own RNA template
2. Telomerase extends unreplicated end.
3. Again, telomerase extends unreplicated end.
Figure Telomerase Prevents Shortening of Telomeres during Replication.
RNA primer
4. Lagging strand is completed.
DNA polymerase
Sliding clamp 27

28. Repairing Mistakes and DNA Damage
DNA replication is very accurate with the average error rate of less than one mistake per billion bases.
DNA polymerase is highly selective:
In matching complementary bases correctly
DNA polymerase inserts the incorrect base
Only about once every 100,000 bases added
Repair enzymes remove defective bases and repair them.
If mistakes remain after synthesis is complete
If DNA is damaged

29. How Does DNA Polymerase Proofread?
DNA polymerase can proofread its work.
It checks the match between paired bases
It can correct mismatched bases when they do occur
If the enzyme finds a mismatch it pauses and removes the mismatched base that was just added.
DNA polymerase III can do this because its e (epsilon) subunit acts as an exonuclease and it removes deoxyribonucleotides from DNA.
This proofreading process reduces the error rate to about 1  10–7 .

30. (a) DNA polymerase adds a mismatched base…
Figure 15.15
(a) DNA polymerase adds a mismatched base…
(b) …but detects the mistake and corrects it.
Figure DNA Polymerase Can Proofread. 30

31. How Does DNA Polymerase Proofread?
If DNA polymerase leaves a mismatched pair behind in the newly synthesized strand a battery of enzymes springs into action to correct the problem.
Mismatch repair occurs when mismatched bases are corrected after DNA synthesis is complete.
Mismatch repair enzymes:
Recognize the mismatched pair
Remove a section of the newly synthesized strand that contains the incorrect base
Fill in the correct bases

32. (a) DNA polymerase adds a mismatched base…
Figure 15.15
(a) DNA polymerase adds a mismatched base…
(b) …but detects the mistake and corrects it.
Figure DNA Polymerase Can Proofread. 32

33. Repairing Damaged DNA
DNA can be broken or altered and various chemicals and types of radiation are the cause of such damage.
UV light can cause thymine dimers to form and these dimers produce a kink in the DNA strand.

34. DNA strand with adjacent thymine bases
Figure 15.16
UV light
Kink
Figure UV Light Damages DNA.
Thymine dimer
DNA strand with adjacent thymine bases
Damaged DNA strand 34

35. Repairing Damaged DNA
The nucleotide excision repair system recognizes such types of damage and enzymes then remove the single-stranded DNA in the damaged section.
The presence of a DNA strand complementary to the damaged strand provides a template for resynthesis of the defective sequences.

36. 3. Nucleotide replacement.
Figure 15.17
Damaged bases
Cut
Cut
1. Error detection.
2. Nucleotide excision.
3. Nucleotide replacement.
Figure In Nucleotide Excision Repair, Defective Bases Are Removed and Replaced.
Repaired damage
4. Nucleotide linkage. 36

37. DNA Repair Genes and Cancer
Defects in the genes required for DNA repair are frequently associated with cancer.
If mutations in the genes involved in the cell cycle go unrepaired.
The cell may begin to grow in an uncontrolled manner
This growth can result in the formation of a tumor
If the overall mutation rate in a cell is elevated because of defects in DNA repair genes then the mutations that trigger cancer become more likely.

38. Please highlight all the terms in blue.
When you are done with the notes:
Read and study chapter 15 in the textbook.
Read and review your notes.
Come prepared for discussion and activities in class.