Ch. 22 Part 2: DNA Mutations and Repair Flashcards
How is genetic stability accomplished?
- Accurate DNA replication system 1/10^6
- DNA repair system when damaged 1/10^8
Define mutation
Any change in genetic material or base sequence of DNA
2 consequences of mutations
Somatic mutations: cell changes (disease/cell death) that mostly affect individual
Germ line mutations: heritable diseases (or stable changes leading to evolution)
Rate of mutation
1 mutation/ 10^9
7x10^9 bp/human genome
7 mutations/cell
100 trillion cells
= A LOT of mutations
Explain the 2 types of mutations
- 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
Explain difference between transition and transversion
Both examples of base substitution
Transition: Purine to purine
Transversion: Purine to pyrimidine (vice versa)
How else can you classify base substitutions?
Based on consequence:
- Silent: Don’t induce change in amino acid
- Missense: change amino acid
- Nonsense: Introduce stop codon (UAA, UAG, UGA)
What is a frameshift mutation?
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)
What is a translocation mutation?
Large scale; Interchange large segments of DNA
Inversion mutation
Large scale; Flip DNA orientation with respect to chromosome (upside down)
Deletion mutation (large scale)
Loss of important genes, can also be caused by chromosomal rearrangment during meiosis (not always bad)
Nondisjunction mutation
Large scale; pairs of chromosomes fail to separate properly
What are insertion, non-frameshift disease?
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)
What do intercalating agents do?
Insertion and cause frameshift (introduce new base pair)
Exon skipping
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.
What is a mutagen?
Physical agent/chemical reagent that causes mutation
Mutagenesis
Process of producing a mutation (induced, spontaneous)
Spontanous mutagenesis
Extensive damage occurs continuously
Faulty editorial proofreading during replication
Chemical mutagenesis
Chemicals from environment (mutagen, carcinogen) induce modification of bases (alkylation) or insertion between bases
UV, Ionizing radiation mutagenesis
Cross linking of base pairs, ring opening, strand breaks, ROS
What is the Ames Test?
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
Explain the Ames Test with salmonella typhirium
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.
What are the 4 different types of DNA damages?
- 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.
- 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)
- Thymine dimers: UV radiation causes 2 adjacent pyrimidine bases to form dimers.
- Chemical modification: ROS formed by oxidative metabolism and ionizing radiation
- Replication errors during replication
- Inter-strand crosslinks by bifunctional alkylating agents, UV, or ionizing radiations
- DNA-protein crosslinks:
- Strand breaks: ionizing radiation can cause single/double strand nicks/breaks
What are the 3 broad categories of repair?
- Direct reversal
- Excision of damaged region, followed by precise replacement
- Damage tolerance: attempt to minimize effects of damage that’s not repaired
Direct reversal of damage
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
Excision Repair (and 3 mechanisms)
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
Mismatch repair MMR
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
Base Excision Repair
Remove incorrect (U) or damaged (3 methylated A) bases
- Removal wrong base by DNA N-glycosylase to create AP site
- Nicking of damaged DNA strand by AP endonuclease upstream of AP site (3’OH terminus next to site)
- Extension of 3’OH terminus by DNA polymerase, DNA ligase seals nick
What repair system is damaged in xeroderma pigmentosum?
Nucleotide excision repair
Nucleotide Excision Reapir
Most important of all the repair systems
- Damage recognition
- Binding of multiprotein complex at damage site (UvrA, B, C, D)
- Double incision of damaged strand SEVERAL NUCLEOTIDES AWAY on both 5’ and 3’
- Removal of damage-containing oligonucleotides in between nicks
- Fill with DNA polymerase
- Ligation via DNA ligase
Damage Tolerance
SOS (Save our Ship) repair
- Cells overwhelmed by UV damage
- DNA polymerases bypass damage site
- Insert DNA without proper base pairing/proofreading (on purpose)
- Recognized by RecA and bind to ss DNA
- Inactivate LexA (repressor of SOS genes)
- Inactivate inhibitor=activate SOS
- DNA polymerase V bypase damaged site (no proofreading)
- Not very accurate
How can you repair DNA ds breaks?
Homologous recombinationa and Homologous end-joining
Homologous Recombination
Rad51 protein searches homologous copy of damaged DNA on sister chromatid and use sequnce from sister chromatid
Non Homologous End joining
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)
Xeroderma Pigmentosum
Mutations in 7 genes XPA-G that encode UV excision system (nucleotide excision repair). Severe light sensitivity, weird pigmentation, neurological defects
Ataxia telangiectasia
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
Hereditary Nonpolyposis colon cancer
Mutations of 1/5 genes hMSH2, hPMS1, MSH6 (mismatch repair) all on chromosome 2
hMLH1 (ch. 3) and hPMS2 (ch. 7)
Faconi’s anemia
aplastic anemia due to defective DNA repair on interstrand cross links or x ray induced DNA damage
Hutchinson-Gilford progeria Syndrome
helicase mutations
“Werner syndrome”
Accelerates aging process 7x normal rate
Bloom’s Syndrome
DNA ligase partially defective (genetic instability -> frequent breaks and changes)
Cockayne’s Syndrome
Profound growth retardation, retinopathy, cataracts, defective UV induced DNA repair system
Retinoblastoma
Autosomal dominant
Mutation in tumor suppressor RB gene on chromosome 13q
