Lectures 3 and 4: Mechanisims of Mutation 1 and 2 Flashcards
(88 cards)
1
Q
What is Human Genetic Variation?
A
Variation in structure or sequence of the human genome
2
Q
Human Genetic Variation AFFECTS WHO?
A
- Can be both within and among populations
1 – Inter-individual (intra-individual)
2 – Inter-population
3
Q
- Multiple mechanisms contributing to Human genetic Variation = 6
A
1- Meiotic recombination
2 – DNA replication and repair
3 – Population effects
…..4 * Random genetic drift
…..5 * Selection (adaptive advantage)
…..6* Migration
4
Q
reference genome ….
A
A reference genome (also known as a reference assembly) is a digital nucleic acid sequence database, assembled by scientists as a representative example of the set of genes in one idealized individual organism of a species.
5
Q
Types of Variation…
STRUCTURAL VS SEQUENCE LEVEL = 6
A
- Structural (>1000bp)
1 – Copy number (deletions & duplications)
2 – Positional (insertions, translocations)
3 – Orientational (inversions) - Sequence level (<1000bp)
4 – Single base substitutions
5 – Small insertions/deletions/duplications
6 – Repetitive sequence
6
Q
Causes of Sequence Variation
A
1 * Homologous DNA recombination during meiosis (allelic and non-allelic)
2 * Retrotransposition
3 * Spontaneous chemical change
4 * Damage due to environmental factors
– Ionising radiation,
– UV radiation
– Chemical mutagens
– Infectious agents (viruses)
5 * Errors of DNA replication and repair
– Proof-reading errors
– Replication slippage
– Replications fork-stalling
7
Q
Causes of Sequence Variation:
DAMAGE DUE TO ENVIRONMENTAL FACTORS = 4
A
1 – Ionising radiation,
2 – UV radiation
3 – Chemical mutagens
4 – Infectious agents (viruses)
8
Q
Causes of Sequence Variation:
Errors of DNA replication and repair: 3
A
1 – Proof-reading errors
2 – Replication slippage
3 – Replications fork-stalling
9
Q
Chemical Stability of DNA: 6
A
1 * DNA is subject to hydrolysis, oxidation, and non-ezymatic methylation ‘in vivo’
2 * Many of these changes interfere with;
…3 – regular base-pairing and/or
…4 – the physical structure of the DNA
5 * The chemical stability of DNA is limited and does play a role in mutagenesis
6 * DNA repair mechanisms counter balance the ongoing change to the genome
10
Q
Nucleotides known to be modified by;
A
- OXIDATIVE DAMAGE
- HYDROLYTIC ATTACK
- UNCONTROLLED METHYLATION
LOOK AT DIAGRAM IN SLIDE 7
11
Q
Tautomers: Spontaneous Change
WHAT IS IT?
A
Tautomers are structural isomers of that readily interconvert with the relocation of a proton.
12
Q
Tautomers: STABLE DNA …TRANSITIONS OCCUR… UNSTABLE TAUTOMERS …
A
- Stable DNA bases exist in:
* Keto form (T and G)
* Amino form (A and C)
2 * Transitions occur to unstable forms:
* Enol (T and G)
* Imino (A and C)
3 * Unstable tautomers can form unstable pairs:
* T:G
* A:C
13
Q
PURINES …PYRIMIDINES …STABLE TO UNSTABLE
A
LOOK AT DIAGRAM SLIDE 8
14
Q
Tautomeric shifts allow
A
Tautomeric shifts allow for irregular base pairing
15
Q
Tautomeric shifts allow for irregular base pairing
WHAT ARE THEY?
A
- Standard base-pairing arrangements
- Anomalous base arrangements
16
Q
Tautomeric shifts allow for irregular base pairing
A
diagram ..understand …on slide 9
17
Q
Replication embeds change
A
- the change caused by the tautomeric shift is embedded by the replication machinery
18
Q
Replication embeds change = 5
A
UNDERSTAND DIAGRAM ON slide 10
- Parental DNA
DNA REPLICATION - …rare enol tautomeric form of guanine (G*)
- First-generation progeny (x2)
- DNA Replication
- SECOND GENERATION PROGENY
- Wild-type
- MUTANT
- Wild-type
-Wild Type
19
Q
Mutagens
A
- agents that cause an increase in the rate of mutation above a spontaneous background (X-rays, UV, chemicals, viruses etc)
20
Q
MUTAGENS:
- Mechanisms Include = 8
A
1 – Deamination (spontaneous and induced)
2 – Alkylation
3 – Depurination
4 – Hydroxylation/Oxidation
5 – Base analogs
6 – Intercalating agents
7 – Ultraviolet radiation
8 – Ionising radiation
21
Q
MUTAGENS = RADIATION EXAMPLES
A
- UV (from sunlight)
- X-rays (medical uses)
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Q
MUTAGENS ..CHEMICAL examples …3
A
- Carcinogens (e.g cigarettes)
- Processed foods and preservatives
- Cosmetics and cleaning products
23
Q
MUTAGENS …INFECTIOUS AGENTS EXAMPLES = 2
A
- Viruses (e.g. HPV)
- Bacteria (e.g. Helicobacter)
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Q
A deaminating agents…
A
is a role played by a chemical agent which exhibits the capability of causing the loss of an amine functional group on another molecular entity (e.g. DNA or protein).
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Mutagens: Deaminating Agents
* Deamination can occur by various means:
-- Spontaneous
– Induced
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Mutagens: Deaminating Agents.....
* Methyl-Cytosine becomes T
* Methyl-Cytosine becomes T
– Spontaneous
– C:G becomes T:A
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Mutagens: Deaminating Agents...
* Effects on Methylation?
HNO2 is a potent deaminator
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Mutagens: Deaminating Agents
LOOK AT SLIDE 12
UNDERSTAND A AND B PROCESSES
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Induced Deamination = 3
1 * Nitrous Acid (HNO2) is a potent driver of oxidative deamination
2 * Hypoxanthine similar to A (imine)
– 'A:T pair becomes G:C'
3 * Xanthine similar to G (enol)
– 'G:C pair becomes A:T'
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Induced Deamination DIAGRAM
UNDERSTAND DIAGRAM ...SLIDE 13
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Understanding Deamination of 5(^m)C in CpG Islands = 7
1 * Predicted that ~4% of the genome should be CpG – reality : 0.8%
2 * Most CpG islands <1800bp long, 60-70% GC (genome average 45-50%)
3 * CpG islands associated with the 5’ end of 40-50% of known genes
4 * Up to 10% of CpG are methylated on the C nucleotide
5 * Methylation can result in repression of expression (tissue-specific methylation
in restricted genes for example)
6* De-amination of the C results in a T nucleotide (so CpG becomes TpG)
7 * Effects of changes variable (from nothing to chronic hemolytic anemia)
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Deamination of 5mC in CpG Islands DIAGRAM
UNDERSTAND SLIDE 14
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Hydroxylamine
* hydroxylates the amino group of cytosine and can lead to G:C to A:T transitions
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Hydroxylamine DIAGRAM
UNDERSTAND SLIDE 15 DIAGRAM
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Mutagens: Alkylating Agents = 4
1 * Chemicals that donate alkyl groups to other molecules.
2 * Cause transitions, transversions, frameshifts, and chromosome aberrations
3 * Alkylation of bases can change base-pairing properties (eg GC to AT)
4 * Alkylation can also activate errors during repair processes
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Alkylating Agents EQUATION
C(n)H(2n+1)
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Alkylating agents ...
Di-(2-chloroethyl) sulfide (mustard gas)
Ethyl methane sulfonate (EMS)
Ethyl ethane sulfonate (EES)
EQUATIONS ...SLIDE 16
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Depurination:
* If not repaired before replicated;
Hydrolysis reactions remove purine (A and G) rings by cleaving the N-glycosidic bond that holds them to the sugar
* If not repaired before replicated;
1 – any base may be added (commonly an A)
2 – position may be (skipped)deleted
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DEPURINATION DIAGRAM...
DIAGRAM ON SLIDE 17
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Mutagens: Intercalating Agent: 5
1 * Thin, plate-like hydrophobic molecules insert themselves between adjacent base pairs
2 * Generally (+) charged molecules
3 * Mutagenic intercalating agents cause insertions during DNA replication.
4 * Loss of intercalating agent can result in deletion.
5 * Examples:
–Proflavin
–ethidium bromide
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Mutagens: Intercalating Agent: MUTATION BY ADDITION = 4
1. Template DNA strand
- Molecule of intercalating agent
2. NEW DNA STRAND
- A randomly chosen base is inserted opposite intercalating agent; here the base is G
3. SUBSEQUENT REPLICATION OF NEW STRAND
4. RESULT: FRAMESHIFT MUTATION DUE TO INSERTION OF OBE BASE PAIR (CG)
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Mutagens: Intercalating Agent: MUTATION BY DELETION = 3
1. Template DNA strand
- Molecule of intercalating agent
2. NEW DNA STRAND
- A randomly chosen base is inserted opposite intercalating agent;
3. Replication of new strand after intercalating agent lost
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Mutagens: Intercalating Agent DIAGRAMS....
UNDERSTAND SLIDE 18
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Mutagens: Base Analogues = 5
1 * Similar structures to regular DNA bases
2 * When incorporated into DNA, they increase frequency of
mis-pairing
........3
– 2-aminopurine : adenine analogue which pairs with cytosine
......4
– 5-bromouracil : thymine analogue pairs with guanine
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Mutagens: Base Analogues DIAGRAM
UNDERSTAND DIAGRAM ON SLIDE 19
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Mutagens: UV Irradiation: 3
1 * UV (254-260 nm) causes purines and pyrimidines to form abnormal dimer bonds and bulges in the DNA strands
2 * Cross-linking of adjacent thymine dimer formation most common
– Block DNA replication and activates DNA repair mechanisms
3.* Hydrolysis of cytosine to a hydrate may lead to mis pairing during replication
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Mutagens: UV Irradiation DIAGRAM
UNDERSTAND DIAGRAMS ON SLIDE 20
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Mutagens: Ionising Radiation = 2
1 * X-rays, gamma rays, alpha and beta particles and neutrons
2 * Can produce double strand breaks, abasic sites and single strand breaks
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Mutagens: Ionising Radiation DIAGRAM
UNDERSTAND DIAGRAMS ON SLIDE 21
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DNA Replication
* DNA polymerase makes errors = 3
1 * DNA polymerase makes errors
2 – Approximately 1/10,000-100,000 nucleotides (10^4 – 10^5)
3 – After repair, approximately 1 in 1 billion (10^9) nucleotides
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DNA Replication
* Two types of abnormalities need repair: 2
1. Base mismatches
2. Damage to the structure of the DNA itself, (eg. breaks in the chromosome or pyrimidine dimers)
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DNA Replication.... mismatch/damage ..HOW IS IT CORRECTED?
Several different types of repair system, and most mismatch/damage can be corrected by more than one system
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Direct DNA Repair = 2
1. SINGLE STRAND BREAKS (ssDNA)
2. ENZYMATIC REPAIR
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Direct DNA Repair
* Single strand breaks (ssDNA) = 3
1. aka DNA “Nicks”
2. DNA Ligase can rejoin backbone
3. Requires functional 5’ phosphate and 3’-hydroxyl groups
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Direct DNA Repair
Enzymatic Repair: 2
Highly specific removal of chemical groups
– e.g. removal of Alkyl group by O6-methylguanine-DNA methyltransferase
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Direct DNA Repair DIAGRAMS
LOOK AND UNDERSTAND SLIDE 24
DIAGRAMS ON BOTH DIRECT DNA REPAIRS
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DNA Repair: Base Excision Repair (BER)...WHAT IS THE PROCESS?
1 - base specific DNA glycosalase remove altered base
2 - Now facing an abasic scenario (i.e. no base)
3 - AP endonuclease removes the sugar back bone
4 - DNA polymerase replaces the missing nucleotide
5 - DNA ligase seals the SSDNA break
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DNA Repair: Base Excision Repair (BER) DIAGRAM
UNDERSTAND THE DIAGRAM AND THE PROCESS ON SLIDE 25
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DNA Repair: Nucleotide Excision Repair (NER) = 4
1. detects and repairs distortions in the DNA helix
2 - Excision nuclease removes nucleotides in and around the distortion
3 - DNA polymerase replaces the missing nucleotides
4 - DNA ligase seals the ssDNA break
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DNA Repair: Nucleotide Excision Repair (NER) DIAGRAM
UNDERSTAND THE DIAGRAM AND THE PROCESS ON SLIDE 26
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DNA Repair: Mismatch Repair (MMR) = 5
1 - Corrects post-replicative base-pair mismatches and insertion/deletion loops
2 - Complex scans DNA for SSB = new strand
3 - Exonuclease removes up to 1000bp of new strand
4 - DNA polymerase replaces the missing nucleotides
5 - DNA ligase seals the ssDNA break
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DNA Repair: Mismatch Repair (MMR) DIAGRAM
UNDERSTAND THE DIAGRAM AND THE PROCESS ON SLIDE 27
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DNA Replication: Slippage
1 * Repeating regions effected, mostly microsatellites
2 * DNA polymerase “slips off” and misaligns with a nearby repeat
3 * Misalignment creates a distortion in the helix which should be detected and repaired by the mismatch repair system
4 * Replication before repair will produce a new allele (expansion more common)
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DNA Replication: Slippage DIAGRAM
UNDERSTAND THE DIAGRAM AND THE PROCESS ON SLIDE 28
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DNA Repair: Homologous Recombination (HR) = 6
1 * ~10 DNA double strand breaks (DSB) per day per cell
2 * Due to radiation, ROS,
broken/stalled replication forks
3 * HR can repair DSBs
....4 * Rare
....5 * High fidelity but not error-free!
6 * Mutations in HR associated genes can be pathogenic
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DNA Repair: Homologous Recombination (HR) DIAGRAM
UNDERSTAND THE DIAGRAM AND THE PROCESS ON SLIDE 29
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DNA Repair: Non-Homologous End-Joining (NHEJ) = 4
1 * Double strand break (DSB) repair
2 * Multiple rounds of resection and addition possible
3 * The process is error-prone around the repair junction
4 * NHEJ can also repair DSB without loss/change
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DNA Repair: Non-Homologous End-Joining (NHEJ) DIAGRAM
UNDERSTAND THE DIAGRAM AND THE PROCESS ON SLIDE 30
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LIST THE WAYS OF DNA REPAIR = 6
1.Non-Homologous End-Joining (NHEJ)
2.Homologous Recombination (HR)
3. Slippage
4.Mismatch Repair (MMR)
5. Nucleotide Excision Repair (NER)
6. Base Excision Repair (BER)
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DNA Repair = 3
GENERALLY SPEAKING
1 * Not every change is repaired before it is replicated
2 * Not every detected change is successfully repaired
3 * The repair genes themselves can be mutated, leading to pathology
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Type of DNA repair = Base excision repair (BER)
...Mechanism..Genes..Disorders..
Base excision repair (BER)
- Removal of abnormal bases
- MYH
- Colorectal cancer
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Type of DNA repair = Nucleotide excision repair (NER)
...Mechanism..Genes..Disorders..
Nucleotide excision repair (NER)
- Removal of thymine dimers and large chemical adducts
- XP genes
- Xeroderma pigmentosum
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Type of DNA repair = Post-replication repair
...Mechanism..Genes..Disorders..
Post-replication repair
- removal of double-strand breaks by homologous recombination or non-homologous end-joining
- NBS, BLM, BRCA1/2
- Nijmegen breakage syndrome
- bloom syndrome
- breast cancer
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Type of DNA repair = MISMATCH REPAIR (MMR)
...Mechanism..Genes..Disorders..
MISMATCH REPAIR (MMR)
- Corrects mismatched bases caused by mistakes in DNA replication
- MSH and MLH genes
- Colorectal cancer (HNPCC)
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HNPCC
hereditary non-polyposis colorectal cancer
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Classifying mutations..
ORIGIN:
SPONTANEOUS AND INDUCED
SPONTANEOUS: Occurs in absence of known mutagen
INDUCED: occurs in presence of known mutagen
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Classifying mutations..
CELL TYPE:
SOMATIC VS GERMLINE
SOMATIC - occurs in non-reproductive cells
GERMLINE: occurs in reproductive cells
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Classifying Mutations... EXPRESSION
CONDITIONAL VS UNCONDITIONAL
CONDITIONAL: Expressed only under restrictive conditions (such as high temperature)
UNCONDITIONAL : expressed under permissive conditions as well as restrictive conditions
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Classifying Mutations...
EFFECT ON FUNCTION =4
1. LOSS OF FUNCTION (KNOCKOUT, NULL) - eliminates normal function
2. HYPOMORPHIC (LEAKY) - reduces normal function
3. HYPERMORPHIC - increases normal function
4. GAIN OF FUNCTION (ECTOPIC EXPRESSION): expressed ar incorrect time in or in appropriate cell types.
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Classifying Mutations...
MOLECULAR CHANGE = 5
1. NUCLEOTIDE SUBSTITUTION = one base pair in duplex DNA replaced with a different base pair
2. TRANSITION = pyrimidine (T OR C) to pyrimidine, or purine ( A or G) to purine
3. TRANSVERSION: pyrimidine (T or C) to purine or purine (A or g) TO PYRIMIDINE
4. INSERTION: One or more extra nucleotides present
5. DELETION: One or more missing nucleotides
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Classifying Mutations... EFFECT ON TRANSLATION = 4
1. SYNONYMOUS (SILENT) = no change in amino acid encoded
2. MISSENSE (NONSYNONYMOUS) = chnage in amino acid encoded
3. NONSENSE (TERMINATION) = Creates translational termination codon (UAA, UAG or UGA)
4. FRAMESHIFT = Shifts triplet reading of codons out of correct phase
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Effects of mutations...
germline, somatic, transition, transversion
1 * Germline mutations
- Present in either (or both) the sperm and egg that made the individual, therefore present in every cell the individual has
2 * Somatic mutations
- Arise after fertilization, during cell replication/division/differentiation/migration, therefore only present in a subset of the individual’s cells
3 * Transition:
- purine-purine or pyrimidine-pyrimidine substitution
4 * Transversion:
- purine-pyrimidine substitution or vice versa
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Effects of mutations: DIAGRAM
LOOK AND UNDERSTAND DIAGRAM IN SLIDE 33
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Effects of mutations...TYPES OF MUTATIONS AND WHAT DO THEY DO...6
1 * Missense mutation:
- causes one amino acid to replace another
2 * Nonsense mutation:
- creates a STOP codon at the site of the mutation
3 * Neutral mutation:
- changes the amino acid content of the protein, but has no functional consequences
4 * Silent (synonymous) mutation:
- does not change the amino acid content of
the protein
5 * Frameshift mutation:
- A mutation that shifts the ribosome’s reading frame, by
inserting or deleting nucleotides in the mRNA
6 * Splice mutation
- A mutation that affects the pattern of RNA splicing, thereby changing the content of the mRNA
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Effects of mutations
..LOSS OF FUNCTION VS GAIN OF FUNCTION...2
1 * Loss-of-function mutations impair the function of the protein
– Multiple ways to do this (reduced expression, function removed)
2 * Gain-of-function mutations cause a protein to perform more of its function
– Over expression or expression at incorrect time/location
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* Effects can vary and are often subtle but are dependent on CONTEXT; 7
1 * Junk DNA (often no effect)
2 * Promoter regions (level of mRNA)
3 * UTRs (tissue distribution, mRNA stability)
4 * Coding regions (Synonymous and non synonymous)
5 * Splice sites (altered splicing)
6 * Introns (splicing enhancers/silencers)
7 * Premature stop codons (truncated proteins)
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“Silent” mutations may not be all they appear....
“Silent” mutations may not be all they appear and may change the splicing of the mRNA (intronic mutations may also)
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How do mutations cause disease?
IMPORTANT TO LEARN THE DIAGRAM = SLIDE 36
