Ch. 22 Part 2: DNA Mutations and Repair

Question 1

How is genetic stability accomplished?

Answer 1

1. Accurate DNA replication system 1/10^6
2. DNA repair system when damaged 1/10^8

Question 2

Define mutation

Answer 2

Any change in genetic material or base sequence of DNA

Question 3

2 consequences of mutations

Answer 3

Somatic mutations: cell changes (disease/cell death) that mostly affect individual

Germ line mutations: heritable diseases (or stable changes leading to evolution)

Question 4

Rate of mutation

Answer 4

1 mutation/ 10^9

7x10^9 bp/human genome

7 mutations/cell

100 trillion cells

= A LOT of mutations

Question 5

Explain the 2 types of mutations

Answer 5

• Small scale: single/few bases change
  
  • i.e. Base substitution (transition vs. transversion), base deletion (frameshift), base insertion
• Large scale: chromosomal mutations
  
  • Translocation, inversion, deletion, nondisjunction

Question 6

Explain difference between transition and transversion

Answer 6

Both examples of base substitution

Transition: Purine to purine

Transversion: Purine to pyrimidine (vice versa)

Question 7

How else can you classify base substitutions?

Answer 7

Based on consequence:

1. Silent: Don’t induce change in amino acid
2. Missense: change amino acid
3. Nonsense: Introduce stop codon (UAA, UAG, UGA)

Question 8

What is a frameshift mutation?

Answer 8

Can be caused by deletion or insertion. Causing misreading of sequence, so entirely new amino acid is given. Not called a frameshift if 3 codons, but can still cause disease (Fragile X, Huntington’s)

Question 9

What is a translocation mutation?

Answer 9

Large scale; Interchange large segments of DNA

Question 10

Inversion mutation

Answer 10

Large scale; Flip DNA orientation with respect to chromosome (upside down)

Question 11

Deletion mutation (large scale)

Answer 11

Loss of important genes, can also be caused by chromosomal rearrangment during meiosis (not always bad)

Question 12

Nondisjunction mutation

Answer 12

Large scale; pairs of chromosomes fail to separate properly

Question 13

What are insertion, non-frameshift disease?

Answer 13

Fragile X: CGG repeat (5-54 unaffected, 60-230 carriers, 230-4000 retarded)

Huntington’s disease: CAG repeat (11-30 unaffected, 36-121 chorea, death)

Question 14

What do intercalating agents do?

Answer 14

Insertion and cause frameshift (introduce new base pair)

Question 15

Exon skipping

Answer 15

base substitution mutation that causes it to no longer think it’s the end of the exon, so includes exon 1, intron, and exon 2 as ONE WHOLE EXON. Essentially, exon 2 skipped.

Question 16

What is a mutagen?

Answer 16

Physical agent/chemical reagent that causes mutation

Question 17

Mutagenesis

Answer 17

Process of producing a mutation (induced, spontaneous)

Question 18

Spontanous mutagenesis

Answer 18

Extensive damage occurs continuously

Faulty editorial proofreading during replication

Question 19

Chemical mutagenesis

Answer 19

Chemicals from environment (mutagen, carcinogen) induce modification of bases (alkylation) or insertion between bases

Question 20

UV, Ionizing radiation mutagenesis

Answer 20

Cross linking of base pairs, ring opening, strand breaks, ROS

Question 21

What is the Ames Test?

Answer 21

Determines if a chemical is a mutagen

Assume: any substance that is a mutagen may also be a carcinogen.

Some substances that cause cancer don’t give positive Ames test (carcinogen, but not mutagen)

Important because low-cost and easy to use

Question 22

Explain the Ames Test with salmonella typhirium

Answer 22

Salmonella typhirium carries mutant gene (can’t make histidine) from ingredients on medium (histidine must be supplied). If back mutation occurs (so have functional gene) and grows on a histidine deficient medium = carcinogen.

Question 23

What are the 4 different types of DNA damages?

Answer 23

1. Depurination or Depyrimidination (Base loss): glycosyl bond linking DNA bases with deoxyribose is labile under physiological conditions. Base lost, but sugar-phosphate backbone remains in tact. If not repaired, strand can’t replicate.
2. Deamination (base modification): A, G, C contain amino groups and can deAMINATE at neutral pH. Esp common from C-> U (easily detected by DNA repair)
3. Thymine dimers: UV radiation causes 2 adjacent pyrimidine bases to form dimers.
4. Chemical modification: ROS formed by oxidative metabolism and ionizing radiation
5. Replication errors during replication
6. Inter-strand crosslinks by bifunctional alkylating agents, UV, or ionizing radiations
7. DNA-protein crosslinks:
8. Strand breaks: ionizing radiation can cause single/double strand nicks/breaks

Question 24

What are the 3 broad categories of repair?

Answer 24

1. Direct reversal
2. Excision of damaged region, followed by precise replacement
3. Damage tolerance: attempt to minimize effects of damage that’s not repaired

Question 25

Direct reversal of damage

Answer 25

Photoreactivation: photolyase uses photon energy 300-600 nm to cleave pyrimidine dimers. Not found in placental mammals MGMT (O6 methyl guanine DNA): suicide enzyme that transfers O6 methyl of bad bases to Cys of itself, rendering it inactive. DNA ligase: sealing nicks

Question 26

Excision Repair (and 3 mechanisms)

Answer 26

If damage in only 1 strand, can cut it out and replace it with new DNA using complementary strand as template All organisms use 3 mechanisms: mismatch, base excision, nucleotide excision

Question 27

Mismatch repair MMR

Answer 27

Correct base mismatches in newly made DNA (unmethylated, unlike parent strand) * Ex in bacteria: If T-G mismatch (T is parent strand) * MutS scans, finds mismatch and binds to base * MutH recognizes methylated sequence on GATC of parent strand * MutL links MutS and MutH * MutH nicks opposite strand (not parent) * Helicase UvrD unwinds DNA from nick, going past nucleotide * Exonucelase excise short segment of "new strand" * SSB binds and DNA pol III fills site * DNA ligase seals In eukaryotes: * MSH2-MSH6=MutS * MLH1, PMS2, and EXO1=MutL * ??=MutH

Question 28

Base Excision Repair

Answer 28

Remove incorrect (U) or damaged (3 methylated A) bases 1. Removal wrong base by DNA N-glycosylase to create AP site 2. Nicking of damaged DNA strand by AP endonuclease upstream of AP site (3'OH terminus next to site) 3. Extension of 3'OH terminus by DNA polymerase, DNA ligase seals nick

Question 29

What repair system is damaged in xeroderma pigmentosum?

Answer 29

Nucleotide excision repair

Question 30

Nucleotide Excision Reapir

Answer 30

Most important of all the repair systems 1. Damage recognition 2. Binding of multiprotein complex at damage site (UvrA, B, C, D) 3. Double incision of damaged strand SEVERAL NUCLEOTIDES AWAY on both 5' and 3' 4. Removal of damage-containing oligonucleotides in between nicks 5. Fill with DNA polymerase 6. Ligation via DNA ligase

Question 31

Damage Tolerance

Answer 31

SOS (Save our Ship) repair 1. Cells overwhelmed by UV damage 2. DNA polymerases bypass damage site 3. Insert DNA without proper base pairing/proofreading (on purpose) 4. Recognized by RecA and bind to ss DNA 5. Inactivate LexA (repressor of SOS genes) 6. Inactivate inhibitor=activate SOS 7. DNA polymerase V bypase damaged site (no proofreading) 8. Not very accurate

Question 32

How can you repair DNA ds breaks?

Answer 32

Homologous recombinationa and Homologous end-joining

Question 33

Homologous Recombination

Answer 33

Rad51 protein searches homologous copy of damaged DNA on sister chromatid and use sequnce from sister chromatid

Question 34

Non Homologous End joining

Answer 34

Doesn't require identical chromatid. Joins 2 ends of broken DNA double helix by DNA ligase IV. protein recognizes ds break and recruit protein kinases. Autophosphorylate themselves to prevent possible nuclease attack and promote ligase reactions (more prone to error)

Question 35

Xeroderma Pigmentosum

Answer 35

Mutations in 7 genes XPA-G that encode UV excision system (nucleotide excision repair). Severe light sensitivity, weird pigmentation, neurological defects

Question 36

Ataxia telangiectasia

Answer 36

1/40,000-1/100.000 worldwide ATM, GADD45, and p53 gene involved Neurological problems in balance, recurrent sinus infections, immune system abnormalities, sensitive to radiation

Question 37

Hereditary Nonpolyposis colon cancer

Answer 37

Mutations of 1/5 genes hMSH2, hPMS1, MSH6 (mismatch repair) all on chromosome 2 hMLH1 (ch. 3) and hPMS2 (ch. 7)

Question 38

Faconi's anemia

Answer 38

aplastic anemia due to defective DNA repair on interstrand cross links or x ray induced DNA damage

Question 39

Hutchinson-Gilford progeria Syndrome

Answer 39

helicase mutations "Werner syndrome" Accelerates aging process 7x normal rate

Question 40

Bloom's Syndrome

Answer 40

DNA ligase partially defective (genetic instability -\> frequent breaks and changes)

Question 41

Cockayne's Syndrome

Answer 41

Profound growth retardation, retinopathy, cataracts, defective UV induced DNA repair system

Question 42

Retinoblastoma

Answer 42

Autosomal dominant Mutation in tumor suppressor RB gene on chromosome 13q