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Molecular Biology > Lecture #4 > Flashcards

Lecture #4 Flashcards

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1
Q

Genetic Mutations Part 1

A

Mutation: a permanent change within the DNA sequence.
- Germ line (gets transferred to offspring) vs. somatic cells (does not get transferred to offspring).

Two types of mutations:
-Transitions (more common): T to C or A to G
Pyrimidine vs Purine
- Transversions: T to G/A or A to C/T
Switch from Prymidine to Purine or vice versa (sign of amunogenic agent)

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2
Q

Genetic Mutations Part 2

A

Nucleotide mutations can lead to protein mutations

  • Silent: no change in amino acid sequence
  • Frame shift: reading frame is changed
  • Missense: change in the amino acid sequence
  • Nonsense: amino acid sequence is changed to a stop codon
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3
Q

Genetic Mutations Part 3

A

Point mutations
- Single nucleotide
- Example?
Small deletions/ additions
- Small regions of DNA sequence
DNA microsatellites
- Used as a genetic marker for mapping
- Regions of di/tri/tetra- nucleotide repeats
- Polymerase “slips”
- Can get progressively worse through each generation

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4
Q

Fragile X syndrome

A

Most common single-gene cause for autism and intellectual handicap in boys.

Gene FMR1 on X-chromosome
- Fragile X mental retardation 1
- X- linked dominant
Expansion of CGG leads to promoter methylation and gene silencing
-5-44 repeats normally
- 200 - 1000 times (methylation of chromosome: activates chromatin compaction).

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5
Q

Sickle Cell Anemia

A

Recessive Autosomal Disorder

Mutation in the B(beta)-globin subunit
- A to T transversion
- Glu to Val mutation (loss of a (-) charge)
- Mutated protein is less soluble than WT
Red blood cells adopt a sickle shape
- Rigid, sticky phenotype
Positive aspect:
- Heterozygotes: resistant to Malaria
Parasites doesn’t grow well in sickle cells

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6
Q

How are mutations acquired?

A

Environmental Factors

  • mutagens: chemicals that increase mutation frequency
  • Radiation: UV, y- rays, X- rays (double strand breaks)

Inherited

Spontaneous

  • Chromosome breaks
  • Polymerase slippage
  • water
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7
Q

How are mutations acquired?

A 
Types of mutations
Alkylation
   -Attachment of methyl/ethyl groups
Oxidation
   -Caused by OH• and H2O2
Deamination 
   -Spontaneous in H2O
    -The amino group on Cytosine is hydrolyzed and is replaced by a carbonyl group; it is now Uracil
Depurination
    -Spontaneous in H2O
    - Hydrolysis of the N-glycosyl linkage; left without a base
    -Missing tooth
Thymine dimer
   -UV exposure
Double Strand Breaks
   -Radiation
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8
Q

How are mutations acquired? Base analogs

A

Types of Chemical Mutagens
Base Analogs
-Similar to nucleotide bases
-Taken-up quickly by the cell and incorporated into the DNA
-5-bromouracil: converts an A:T to a G:C

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9
Q

How are mutations acquired? Intercalating Agents

A 
Types of Chemical Mutagens
Intercalating Agents
  -Chemicals that slip between the bases within the double helix and interfere with the base pair stacking
   -Shifts the DNA sequence
   -Ethidium Bromide; Acridine (dye)
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10
Q

How do cells fix the problem?

A

DNA Polymerase
Proofreading capabilities
1 in 107 base pairs has a mistake

Mechanisms that depend on a non-affected strand

  • Repair by Reversal
  • DNA Mismatch Repair
    • 1 in 109 to 1010 base pairs has a mistake
      - Catches most of the mistakes that the Polymerase doesn’t catch
  • Base Excision Repair
  • Nucleotide Excision Repair

Translesion DNA Synthesis

Non-Homologous End Joining

Homologous Recombination

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11
Q

Mechanism Requiring a Non- affected Strand: Repair by Reversal

A

Photo-reactivation

  • In bacteria and plants; humans don’t use this method
    - Energy received from light
    • Reverses the formation of pyrimidine dimers
      • DNA photolyase
      • λ > 300nm (Sunlight and fluorescent light
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12
Q

Mechanism Requiring a Non- affected Strand: Basic Mechanism

A
  • Recognition and removal of the mismatch by various nucleases
    • Repair DNA Polymerase binds and makes a complimentary strand
    • DNA strands are sealed by DNA Ligase (ATP dependent)

Proteins vary for each process

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13
Q

DNA Mismatch Repair (MMR)

A 
E. coli
- MutS recognizes the mismatch due to a distortion in the helix
- MutL is recruited and activates MutH
- MutH binds un-methylated GATC sequence; nicks the DNA strand
- UvrD helicase unwinds the DNA to the mismatch
- Exonucleases
      5`→3` and 3`→5`
      Chews DNA to the mismatch
- DNA Polymerase III
   - Fills in the sequence gap
- DNA Ligase
    Seals the gap

Parental strand is methylated by Dam methylase
- New strand has not been methylated yet

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14
Q

DNA Mismatch Repair (MMR): Eukaryotes

A

Eukaryotes

  • Similar to bacteria but with MANY more proteins
  • MSH proteins
    - MutS homologue
  • MLH proteins
    • MutL homologue
    • PMS
  • No known MutH homologue
  • Exo1: exonuclease

New strand recognized by nicks?
- PCNA: Sliding clamp
Interacts with MSH proteins

Mutations in MSH and MSL proteins lead to hereditary nonpolyposis colorectal cancer
- Predisposition to colon cancer

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15
Q

Base Excision Repair

A

Removes small lesions within the DNA sequence
- Non-helix distorting

Types of mutations

  • Hydrolysis, oxidation, alkylation, deamination, and depurinatiation/depyrimidination
    - Bases or the backbone
Mechanism:
DNA glycosylase recognizes damage
     - Legion specific
AP endonuclease and phosphodiesterase remove the nucleotide
      - Apurinic/apyrimidinic

DNA polymerase fills in the gap

DNA ligase seals the gap

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16
Q

Nucleotide Excision Repair

A

“Fixes” lesions by removing a stretch of nucleotides within the affected strand

  • 12-13 nt in bacteria
    - 24-32 nt in eukaryotes

Removes damage caused by UV light and carcinogens
- Bulky DNA adducts like Thymidine dimers

  • E. coli*
    • UvrA+B: recognition
    • UvrB+C: Melting & Incision
    • Rest is similar to MMR
17
Q

Nucleotide Excision Repair: Eukaryotic

A

Eukaryotic

  • Similar to E. coli
  • XPC detects lesions
  • XPA and RPA facilitate specific recognition
  • TFIIH
    - XPB and XPD act as helicases
  • XPG: endonuclease (3`)
  • ERCC1-XPF endonuclease (5`)
  • PCNA, RPA, Ligase, RFC, Pol fill in the gap

Xeroderma pigmentosum

  • Autosomal recessive
  • Exposure to sunlight results in lesions and cancer
  • Mutations in XPA, XPC, XPD, XPF, XPG
18
Q

Translesion DNA Synthesis

A

What’s worse: Incomplete replication or a mutation?

  • Failsafe method allowing completion replication completion
  • Last resort; not ideal

DNA lesion remains in the parent strand and errors are produced in the daughter strand
- Gives the cell another chance to “fix” the damage

In E. coli:  Part of the SOS response
   - DNA damage response process
   - Activated by RecA
       Involved in homologous recombination
       Binds to ssDNA
   - DNA Pol IV or V used
19
Q

Translesion DNA Synthesis

A

Eukaryotes

  • PCNA (Sliding Clamp) is ubiquitinated when a lesion is encountered
  • Pol δ is switched out for Pol η
    • Nucleotide is incorporated independently of base pairing
    • Specific lesions result in the addition of specific nucleotides
      • T-T yields A A
    • Low processivity
  • Pol η is switched out for Pol δ
20
Q

Non-Homolgous End Joining (NHEJ)

A
  • Used when the DNA helix suffers a double strand break
  • Ends of DNA are ligated together without the need for homologous regions
  • Results in a loss of some genetic information
    • Which is better: lose some information or lose an entire chromosome?

Incorrect NHEJ can lead to translocations and telomere fusion

  • Movement of DNA sequence from one chromosome to another
  • Hallmark of cancer
21
Q

Non-Homologous End Joining (NHEJ)

A
  • Short stretches of DNA can misalign after double strand break
  • Nucleases remove the single strand tails
  • Polymerase and Ligase fill in the gap

Specifically
- Ku70,80 bind the DNA ends and recruit PKc (kinase)
- Complex formed with Artemis
Exonuclease 5→3
Endonuclease activated by phosphorylation

22
Q

Homolgous Recombination

A
  • Exchange of information between a pair of homologous DNA molecules
  • Used to ensure genetic variation
    Meiosis
    New combination of genes; can change the activation of a gene

Opportunity to retrieve sequences lost through DNA damage
Double Strand Breaks (radiation, nicks, lesions)
Very common mechanism

  • Requires the presence of extensive regions of sequence similarity
    Close but not perfect; i.e. alleles; ~ 100bp needed
  • Occurs shortly after replication but before division
    In the case of double strand breaks
23
Q

Homologous Recombination

A
  • Alignment of homologous DNA molecules
  • Introduction of breaks in DNA
    Ends are processes to make ssDNA
  • Strand Invasion
    Single stranded DNA pairs with complimentary strand in homologous DNA molecule
  • Formation of Holiday Junction
    DNA strands cross and branch migration
  • Resolution of Holiday Junctions
    Regenerate DNA and finish genetic exchange through cleavage or dissolution

Molecular Biology (8 decks)

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