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What is the Heredity Material?. Chromosome existence had been known since the late 1800'sMendel completed extensive work on heredityThe nature of Mendel's heredity factors remained a mystery until 1940's. DNA vs. Protein. It was also known that chromosomes were a compilation of DNA and proteinsSo what was this
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1. DNA Replication & Repair Biology 11 Chapter 14
2. What is the Heredity Material? Chromosome existence had been known since the late 1800s
Mendel completed extensive work on heredity
The nature of Mendels heredity factors remained a mystery until 1940s
3. DNA vs. Protein It was also known that chromosomes were a compilation of DNA and proteins
So what was this transmissible factor that was inherited from generation to generation
DNA
Protein
The prevailing theory of the day was that the transmissible factor was protein
4. Why Protein Proteins have almost a limitless variation in structure and function
With all of the different proteins it was reasonable to assume that a significant level of biological diversity could be reached
DNA on the other hand was a simple chemical structure (the actual secondary structure was not known until 1950s)
Only four bases (nucleotides)
A, T, G & C
5. DNA as the Hereditary Material The next slides will outline a series of classical experiments which conclusively showed that DNA and not proteins is the hereditary material
Frederick Griffith 1928s
Oswald Avery 1940s
Hershey Chase 1940s
Watson Crick 1950s
6. Griffith In 1928 Griffith reported a mysterious phenomenon
Transformation
At the time Griffith was working on a vaccine for Streptococcus pneumoniae
Griffith was working with two strains
R rough strain avirulent
S smooth strain virulent
7. Griffith
8. Griffiths Experiment
9. Averys Experiment Began with a normal S type cell
Exposed to heat and other treatments to yield an extract of only
DNA, RNA, & Protein
10. Averys Experiment
11. Critics of Averys Experiment Even after Averys experiment most scientist still believed that protein was the transforming factor
These scientists claimed that the enzymatic treatments were not sufficient to remove all of the proteins
More studies were performed with viruses
12. Hershey Chase They wanted to conclusively answer the question of whether the transforming factor was DNA or protein
Used the T2 bacteriophage virus and E. coli
What is a bacteriophage?
What is a virus?
All viruses are acellular entities which are made up of a protein coat which surrounds the genome
Some viruses can have an envelope (cell membrane) around the protein coat
13. Bacteriophage A bacteriophage is a bacteria eating virus
Many types of these viruses exist
Inject viral DNA into host cells and cause the host cell to make many copies of the virus
14. Bacteriophage Life Cycle The life cycle is as follows
Docking
Adsorption
Replication
It is important to remember that the virus injects only DNA and that it then uses the cellular machinery of the bacterial cell to make more copies of its genome and the proteins
More copies of viral genome
More copies of viral proteins
Assembly
Release
16. Other Viruses It uses its genetic information (DNA or RNA) and directs a cell to make more copies of the virus
HIV
Herpes
Hepatitis
Small Pox
HPV
17. Hershey-Chase Are genes made of DNA or protein
Used a T2 virus that only infects E. coli cells
What was known
T2 injects genes into bacteria
T2 is made up of only DNA & protein
18. Hershey-Chase Using radioactive labels these researches tagged either the DNA or protein population
P32
Why does this tag only DNA
S35
Why does this tag only proteins
19. Hershey & Chase
20. Hershey & Chase
22. Hershey & Chase Conclusion
The genetic material that redirects the functions of an E. coli cell is DNA
After these results were published proponents of the protein hypothesis had to admit that DNA, not protein, must be the hereditary material
23. Watson & Crick In the 1950s Watson and Crick proposed their model for DNA secondary structure
The structure provided rules or directions for how to copy itself provided one of the strands was acting as a template
24. Meselson Stahl Experiment When replication occurs during S phase is the new DNA old and new
Or do the parent strands serve as templates and then reanneal to one another
Conservative vs. Semi conservation Replication
25. Meselson Stahl Experiment To answer this question Meselson and Stahl exploited the differences between N14 and N15
N15 is a heavy isotope with one extra neutron
The difference in mass creates a difference in density
This difference in density can be seen when centrifugation techniques are employed
28. Mechanisms For Replication The mechanism for DNA replication are complex and have only been known for the last 25 years
The key discovery insight began with the discovery of DNA polymerase
DNA polymerases
Catalyze the formation of the daughter strand
There are many types of polymerases enzymes
These enzymes only work in the 5 to 3 direction
29. Initiation of DNA replication Step 1 opening the helix
Proteins bind to specific DNA sequences known as origins of replication
Bacteria have one
Eukaryotes have thousands
AT rich regions
Why?
Helicases aid in the opening of the helix
30. Initiation of DNA replication Role of SSBP
Single stranded binding proteins
After the helix has opened it is prevented from re-annealing by the action of these proteins
These proteins stabilize single stranded DNA
31. Initiation of DNA replication Step 2 binding of RNA primers
Primase adds short stretches of RNA primers
Purpose is to give DNA polymerase a 3OH group from which to add new DNA nucleotides
Two primers are put down as the replication bubble opens
32. Replication Elongation Step 3
After the primers are in place an enzyme known as DNA polymerase III (bacteria; eukaryotes has a similar polymerase but it has a different name) will add new nucleotides to the daughter strand as directed by the template strand
Replication must proceed in the 5 to 3 direction
33. DNA Polymerase
34. Chemistry of DNA Polymerase
38. Replicating Linear Chromosomal Ends What is the chance that primase would add a primer exactly on the last base of the lagging strand?
NOT LIKELY
40. How To Correct Lagging Strand End Shortening Linear chromosome have ends known as telomeres
Telomeres do not contain genes that code for proteins
Telomers are stretches of repetitive bases repeated over and over again
TTAGGG in humans repeated thousands of times
41. How To Correct Lagging Strand End Shortening Telomerase is an enzyme synthesizes DNA from an RNA template contained in the active site of the enzyme
Telomerase binds to the ends of linear DNA as adds these repetitive bases
Telomerase then lengthen the overhang on the lagging strand
42. Telomerase
43. Telomerase Complications Telomerase is not active in most somatic cells
This raises an interesting questions
If telomerase is not active then it is reasonable to assume that cells must be in G0 and not growing and dividing
If telomerase is active then would it lead to uncontrolled cell growth and cancer
44. Telomerase Complications Conflicting results
Knockout mice which do not have telomerase develop tumors
Suggests that telomerase activity is not necessary for tumor formation
But many cancerous cells in humans have telomerase activity which allows then to continue to grow and divide
Adding active telomerase to cultured cells allows them to grow past a normal life span
45. Risks To DNA Replication Synthesizing DNA is risky business
As it turns out DNA polymerase can make mistakes in matching complementary DNA
What happens if during replication DNA mismatches, such as G=T were not corrected?
Mutation would occur at such a rapid rate life itself would not be possible
46. Risks To DNA Replication DNA polymerase inserts the incorrect base once in every 100,000 bases
Error rate of 1 x 10-5
At this rate your genome would be riddle with mutations
But as it turns out DNA polymerase can proofread
47. Proofreading DNA polymerase contains a subunit which is capable of excising a nucleotide in the 3 to 5 direction
Known as a 3 to 5 exonuclease
This reduces the error to one mistake per 10 million bases
Error rate of 1 x 10-7
48. Proofreading
49. Proofreading Mechanism Explains 5 to 3 Replication Requirement In terms of chemistry an incoming 3OH could just as easily attack a 5 triphosphate group
So why then did all DNA replication evolve to synthesize DNA in the 5 to 3 direction?
50. Proofreading Is this error rate of 10-7 acceptable?
NO!!
During or shortly after DNA replication a system known as mismatch repair is active
The enzymes involved in mismatch repair were discovered using E. coli as the model system
MutS, MutL, MutH & others
Humans have MLH, MSH etc.
Humans with defective MMR genes are genetically predisposed to HPNCC
51. Mismatch Repair in E. Coli
52. DNA Damage Even after DNA is synthesized and proofread and mismatches repaired, all is not well
DNA is under constant assault
Spontaneous DNA damage
Breathing
Spontaneous deamination of C
Physical DNA damage
UV light and radiation
Chemical DNA damage
Benzo a pyrene
Nitrates and nitrites
Aflatoxin B1 moldy peanuts and corn
53. Spontaneous DNA Damage Oxidative Damage
Aerobic Respiration causes DNA damage everyday
Reactive oxygen radicals are created from ETC
Deamination Reactions C become U
54. Deamination of Cytosine Spontaneous non-enzymatic deamination of cytosine occurs at a rate of 10-7 cytosine per day
Equal to 100 cytosine to uracil conversions each day
Based on this information why does DNA contain thymine and not uracil?
55. C to U Transversions
56. Physical Damage Ionizing radiation leads to DNA double strand breaks
Cause deletions of large sections of DNA
Gene amplification events
Translocation events part of one chromosome attaches to another chromosome
UV light causes a specific type of mutation
57. Repairof Thymine Dimers
58. Xeroderma Pigmentosum Rare autosomal recessive disease in humans
Extreme sensitivity to UV light
Skin develops lesions even after short exposure
59. Chemical Damage Alkalization of DNA is common when exposed to certain chemicals
Alkalization interferes with proper base pairing and could lead to increased mistakes during replication
Damaged bases can be repaired using BER
60. DNA Repair All cells have repair systems in place to deal with DNA damage
Mismatch repair - MMR
Base excision repair - BER
Nucleotide excision repair - NER
Direct repair
Others