Exam: Cycle 3

Repair of Damage in DNA

1. Not all changes to DNA are mutations: mutations are double-stranded changes
2. Changes in only one strand are DNA damage: includes replication errors resulting in wrong base pairs, chemical reactions that add chemical groups to bases
3. 3 types of repair mechanisms:
4. Proofreading: for errors by DNA polymerase
5. Mismatch repair: for errors made during replication that escape proofreading
6. Excision repair: for various kinds of DNA damage (chemicals, radiation, etc)
7. Correction of errors: recognize error and remove it, replace removed DNA using a repair DNA repair polymerase, seal new DNA to old DNA using DNA ligase
8. Base pair mismatch corrected by a proof-reading mechanism in DNA polymerase during replication, or a DNA repair mechanism after replication
9. DNA polymerase: makes very few errors, usually base-pair mismatches
10. Has a proofreading mechanism: if nucleotide is mismatched, it can use a built-in 3’ to 5’ exonuclease activity to erase the mistake, and it can then continue
11. Major DNA polymerases of replication proofread their work 
12. E. coli’s PolIII is fully functional → error rate of 1 in 1 million
13. Proofreading function inhibited → error rate of 1 in 1000-10,000
14. Similar results for eukaryotes
15. Mismatch repair mechanism corrects about 99% of the 1 in 107 errors left by DNA polymerase → only leaves 1 in 109 errors
16. Evolutionary conservation in bacteria, yeast, humans, etc → ancient, vital
17. Important to prevent cancer
18. A type of colon cancer is caused by a mutation in a gene for a mismatch repair protein
19. Excision repair mechanisms: correct various types of damage
20. Non-bulky damage: may occur in chemical modification of bases, or the loss of purine bases
21. Base-excision repair: removes erroneous base and replaces it with the correct one
22. After proofreading, is the most important correcting mechanism
23. Bulky distortions: due to UV light (causes thymine dimers to form: adjacent T’s bond)
24. Can have serious consequences bc DNA polymerase can’t continue synthesis past the distortion → cell death
25. Nucleotide-excision repair: similar mechanism to other repairs; specific proteins recognize the bulky distortion and remove part of it containing the thymine dimer → repair DNA polymerase and DNA ligase replace and seal it to the rest of the DNA
26. Many other kinds of bulky and non-bulky damage; are repaired as well 
27. After proofreading and repair, there are very few mismatches (will be mutations/double-stranded changes)

Origin of Variation

1. Last replication bubble at the end of chromosomes (only applies to linear DNA, not in circular DNA like in bacteria): shortening of new strand
2. Primer is removed at 5’ end, so there’s a 3’ end sticking out. Can’t add more because there’s no template to add a primer and then fill with nucleotides
3. Newly synthesized strand starts to decrease in length, will eventually affect genes
4. Cells evolved to protect genes from shortening due to DNA replication: telomeres
5. Repeat of TTAGGG: buffers ends of chromosomes (doesn’t code for anything), but isn’t endless
6. Cell senescence (irreversible cell cycle arrest) occurs when cell reaches its Hayflick limit (# of times a cell divides before cell division stops)
7. Cancer cells, germ cells, stem cells keep replicating bc they have active telomerase
8. Telomerase restores length of telomeres; we need to restore the 5’ end  
9. Telomerase will extend the 3’ end to make more template for the replisome to act (primer, PolIII, etc)
10. Telomerase brings its own RNA template to make the DNA template strand 
11. Telomerase always acts in lagging strand
12. Mechanisms to ensure inheritance of sameness (prevent mistakes - but note that mutations aren’t always bad)
13. Proofreading by DNA Polymerase during S phase: for base pairs that are a mismatch 
14. Excision repair enzymes when proofreading fails: detects mistake, cuts newly synthesized strand and removes bad piece, and then DNA polymerase III can resynthesize from 3’ hang and ligase will seal gap (at 3' end of new strand) 
15. Our molecules are designed to ensure sameness
16. Complementary base pairing means that we can check for errors using shape
17. Semi-conservative replication means that DNA polymerase needs a template
18. Sources of DNA damage: 
19. Exogenous factors (from outside cell): UV light, chemicals, ionizing radiation (gamma rays, x-rays)
20. Endogenous factors (from inside cell): DNA replication errors, mitochondria (produce reactive oxygen species [ROS]: produce free radicals, highly reactive)
21. DNA Polymerase III makes a mistake 1 in 106, would be 1 in 1000 if no proofreading: but 3.2 B base pairs, so 3000+ errors in each
22. Mutation is a double-stranded change (one-sided change is just damage)
23. After DNA damage (e.g. wrong base pair), the second round of replication can result in a point mutation when changed base pair gets its complement
24. Thymine dimer: two adjacent T form covalent bonds when UV light absorbed
25. Can be repaired by photolyase (an enzyme) and white light

Doesn't work in humans anymore

1. Humans use excision repair enzymes (lost photolyase through evolution)
2. Melanoma: mutation in repair enzyme, so thymine dimers accumulate to cause skin cancer
3. Double-strand break: repair is during G1 during non-homologous end joining by ligase (just joins ends), may lose nucleotides that fell off, can result in mutation
4. Human Genome Project: sequence of the entire genome
5. 2000: Working draft compiled of DNA from several anonymous donors
6. Mid 2000’s: Individual sequences of Craig Ventor and James Watson (cost $100M at the time)
7. 2010: Individual sequences of Han Chinese, Korean, Yoruba, Bantu (Desmond Tutu), Neanderthal, unborn fetus, etc
8. 2015: 1000 Genomes Project reported
9. People thought they could find the cure to everything from the genome, but the genome was sequenced to do better science more efficiently (look at DNA as a whole and not as individual genes)
10. Goal is to figure out role of DNA in actual diseases
11. DNA sequence: 
12. 25% unknown (probably junk)
13. 10% essential (2% code for proteins)
14. 10% intron (junk)
15. 55% transposons, viruses, and “dead genes” (junk)

Genetic Variation due to Mutation

1. Mutations are not always bad, it leads to evolution
2. Type of mutation is one thing, but location is what affects whether or not there’s a change that affects the organism
3. Ways to get genetic diversity: independent assortment, recombination, mutation
4. Human variation: took 2504 genomes from 26 diff populations, and 0.14% was found to be different from the reference genome (made based on all of them)
5. Chimps have a 1.4% difference
6. Vast majority of variations between people are SNPs (single nucleotide polymorphisms)
7. This is what’s analyzed in DNA analysis: look at specific areas for differences
8. There are abt 12000 variants in coding regions, and abt 100 de novo (not in parents) variants, abt 30 variants are associated with diseases 
9. Mutations are more complicated than just good = helpful, bad = disease
10. Some types of mutations: substitution (point mutation), deletion, insertion, inversion (backwards)
11. Result of point mutations: silent (no change), nonsense (stop code), missense (change in amino acid: conservative if similar structure, nonconservative if not similar structure)
12. Single nucleotide polymorphisms (SNPs): occur in pairs (bc it’s a mutation), most common type of genetic variation among people, can occur anywhere in genome (usually between genes), most have no effect on health/development
13. InDel mutations (insertion/deletion): due to DNA Polymerase slippage, not SNPs
14. Insertion: slippage of new strand, repetitive sequences more prone to slippage
15. Deletion: slippage of template strand, so new strand has one less set of 3 bases
16. Tautomers: spontaneous tautomeric shifts (tautomerization) change base pairing partners
17. Common forms: A and T are normally in keto form, C and G are normally in amino form
18. Rare forms: A/T can switch to enol form, and C and G can switch to imino form
19. They switch partners: not a mismatch; the structures line up so repair enzymes don’t recognize the change
20. If a rare form of a nucleotide comes and pairs in the replication of a parental DNA, the first mutant shows up in second generation of parental DNA (bc tautomerization switches and goes back to normal form in the next round of replication)
21. Mismatch repair can introduce a mutation
22. Can happen if during the repair process, there is tautomerization (so it favours non-Watson/Crick pairing)
23. Can happen bc repair enzymes can cut template strand instead of newly synthesized strand instead 
24. Some mutagens are tautomerically unstable base “analogues” 
25. Ex. 5-bromouracil looks like thymine (has a Br instead of a CH3 on 5’ carbon)
26. 5BU gets put instead of T (is in keto form and favours A), but switches to enol form and favours G
27. After second round of replication, there are two normal strands, one with a mutagen/damage (not a mutation bc it still matches, but may undergo apoptosis), and one with a transition mutation (purine → purine)
28. Transition mutation: purine to purine and pyrimidine to pyrimidine
29. Transversion mutation: purine to pyrimidine or pyrimidine to purine 
30. Transposable elements (jumping genes): regions of DNA that move around
31. Discovered by Barbara McClintock in 1940s in maize: no one believed her, thought genome is stable (and she was a woman)
32. Kernels were spotted bc a TE (transposable element) came in the genes for pigmentation in some cells so pigmentation wouldn’t occur
33. Found in Drosophilia in 1950s, bacteria in 1960s, and humans in 1970s
34. Contain a region that codes for transposase enzyme (does the cutting/copying and pasting)
35. (Don’t need to know about diff types of TEs and mechanism of transposition)
36. Most common in plants, which is why their genomes are so big
37. 85% of barley’s genome is transposable elements
38. 50% of human genome has TEs, 70-80% of human genes contain TEs (up to 1000 per gene)
39. Most TEs don’t move due to inactivating mutations
40. Active TEs have evolved to be in “safe havens” where they don’t interfere with cell’s actions

We also have a mechanism to silence/inactivate TEs, if it doesn’t work → cancer