Lectures 3 and 4: Mechanisims of Mutation 1 and 2 Flashcards
What is Human Genetic Variation?
Variation in structure or sequence of the human genome
Human Genetic Variation AFFECTS WHO?
- Can be both within and among populations
1 – Inter-individual (intra-individual)
2 – Inter-population
- Multiple mechanisms contributing to Human genetic Variation = 6
1- Meiotic recombination
2 – DNA replication and repair
3 – Population effects
…..4 * Random genetic drift
…..5 * Selection (adaptive advantage)
…..6* Migration
reference genome ….
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.
Types of Variation…
STRUCTURAL VS SEQUENCE LEVEL = 6
- 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
Causes of Sequence Variation
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
Causes of Sequence Variation:
DAMAGE DUE TO ENVIRONMENTAL FACTORS = 4
1 – Ionising radiation,
2 – UV radiation
3 – Chemical mutagens
4 – Infectious agents (viruses)
Causes of Sequence Variation:
Errors of DNA replication and repair: 3
1 – Proof-reading errors
2 – Replication slippage
3 – Replications fork-stalling
Chemical Stability of DNA: 6
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
Nucleotides known to be modified by;
- OXIDATIVE DAMAGE
- HYDROLYTIC ATTACK
- UNCONTROLLED METHYLATION
LOOK AT DIAGRAM IN SLIDE 7
Tautomers: Spontaneous Change
WHAT IS IT?
Tautomers are structural isomers of that readily interconvert with the relocation of a proton.
Tautomers: STABLE DNA …TRANSITIONS OCCUR… UNSTABLE TAUTOMERS …
- 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
PURINES …PYRIMIDINES …STABLE TO UNSTABLE
LOOK AT DIAGRAM SLIDE 8
Tautomeric shifts allow
Tautomeric shifts allow for irregular base pairing
Tautomeric shifts allow for irregular base pairing
WHAT ARE THEY?
- Standard base-pairing arrangements
- Anomalous base arrangements
Tautomeric shifts allow for irregular base pairing
diagram ..understand …on slide 9
Replication embeds change
- the change caused by the tautomeric shift is embedded by the replication machinery
Replication embeds change = 5
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
Mutagens
- agents that cause an increase in the rate of mutation above a spontaneous background (X-rays, UV, chemicals, viruses etc)
MUTAGENS:
- Mechanisms Include = 8
1 – Deamination (spontaneous and induced)
2 – Alkylation
3 – Depurination
4 – Hydroxylation/Oxidation
5 – Base analogs
6 – Intercalating agents
7 – Ultraviolet radiation
8 – Ionising radiation
MUTAGENS = RADIATION EXAMPLES
- UV (from sunlight)
- X-rays (medical uses)
MUTAGENS ..CHEMICAL examples …3
- Carcinogens (e.g cigarettes)
- Processed foods and preservatives
- Cosmetics and cleaning products
MUTAGENS …INFECTIOUS AGENTS EXAMPLES = 2
- Viruses (e.g. HPV)
- Bacteria (e.g. Helicobacter)
A deaminating agents…
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).
Mutagens: Deaminating Agents
- Deamination can occur by various means:
– Spontaneous
– Induced
Mutagens: Deaminating Agents…..
- Methyl-Cytosine becomes T
- Methyl-Cytosine becomes T
– Spontaneous
– C:G becomes T:A
Mutagens: Deaminating Agents…
- Effects on Methylation?
HNO2 is a potent deaminator
Mutagens: Deaminating Agents
LOOK AT SLIDE 12
UNDERSTAND A AND B PROCESSES
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’
Induced Deamination DIAGRAM
UNDERSTAND DIAGRAM …SLIDE 13
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)
Deamination of 5mC in CpG Islands DIAGRAM
UNDERSTAND SLIDE 14
Hydroxylamine
- hydroxylates the amino group of cytosine and can lead to G:C to A:T transitions
Hydroxylamine DIAGRAM
UNDERSTAND SLIDE 15 DIAGRAM
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
Alkylating Agents EQUATION
C(n)H(2n+1)
Alkylating agents …
Di-(2-chloroethyl) sulfide (mustard gas)
Ethyl methane sulfonate (EMS)
Ethyl ethane sulfonate (EES)
EQUATIONS …SLIDE 16
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
DEPURINATION DIAGRAM…
DIAGRAM ON SLIDE 17
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
Mutagens: Intercalating Agent: MUTATION BY ADDITION = 4
- Template DNA strand
- Molecule of intercalating agent - NEW DNA STRAND
- A randomly chosen base is inserted opposite intercalating agent; here the base is G - SUBSEQUENT REPLICATION OF NEW STRAND
- RESULT: FRAMESHIFT MUTATION DUE TO INSERTION OF OBE BASE PAIR (CG)
Mutagens: Intercalating Agent: MUTATION BY DELETION = 3
- Template DNA strand
- Molecule of intercalating agent - NEW DNA STRAND
- A randomly chosen base is inserted opposite intercalating agent; - Replication of new strand after intercalating agent lost
Mutagens: Intercalating Agent DIAGRAMS….
UNDERSTAND SLIDE 18
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
Mutagens: Base Analogues DIAGRAM
UNDERSTAND DIAGRAM ON SLIDE 19
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
Mutagens: UV Irradiation DIAGRAM
UNDERSTAND DIAGRAMS ON SLIDE 20
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
Mutagens: Ionising Radiation DIAGRAM
UNDERSTAND DIAGRAMS ON SLIDE 21
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
DNA Replication
- Two types of abnormalities need repair: 2
- Base mismatches
- Damage to the structure of the DNA itself, (eg. breaks in the chromosome or pyrimidine dimers)
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
Direct DNA Repair = 2
- SINGLE STRAND BREAKS (ssDNA)
- ENZYMATIC REPAIR
Direct DNA Repair
- Single strand breaks (ssDNA) = 3
- aka DNA “Nicks”
- DNA Ligase can rejoin backbone
- Requires functional 5’ phosphate and 3’-hydroxyl groups
Direct DNA Repair
Enzymatic Repair: 2
Highly specific removal of chemical groups
– e.g. removal of Alkyl group by O6-methylguanine-DNA methyltransferase
Direct DNA Repair DIAGRAMS
LOOK AND UNDERSTAND SLIDE 24
DIAGRAMS ON BOTH DIRECT DNA REPAIRS
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
DNA Repair: Base Excision Repair (BER) DIAGRAM
UNDERSTAND THE DIAGRAM AND THE PROCESS ON SLIDE 25
DNA Repair: Nucleotide Excision Repair (NER) = 4
- 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
DNA Repair: Nucleotide Excision Repair (NER) DIAGRAM
UNDERSTAND THE DIAGRAM AND THE PROCESS ON SLIDE 26
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
DNA Repair: Mismatch Repair (MMR) DIAGRAM
UNDERSTAND THE DIAGRAM AND THE PROCESS ON SLIDE 27
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)
DNA Replication: Slippage DIAGRAM
UNDERSTAND THE DIAGRAM AND THE PROCESS ON SLIDE 28
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
DNA Repair: Homologous Recombination (HR) DIAGRAM
UNDERSTAND THE DIAGRAM AND THE PROCESS ON SLIDE 29
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
DNA Repair: Non-Homologous End-Joining (NHEJ) DIAGRAM
UNDERSTAND THE DIAGRAM AND THE PROCESS ON SLIDE 30
LIST THE WAYS OF DNA REPAIR = 6
1.Non-Homologous End-Joining (NHEJ)
2.Homologous Recombination (HR)
- Slippage
4.Mismatch Repair (MMR)
- Nucleotide Excision Repair (NER)
- Base Excision Repair (BER)
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
Type of DNA repair = Base excision repair (BER)
…Mechanism..Genes..Disorders..
Base excision repair (BER)
- Removal of abnormal bases
- MYH
- Colorectal cancer
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
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
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)
HNPCC
hereditary non-polyposis colorectal cancer
Classifying mutations..
ORIGIN:
SPONTANEOUS AND INDUCED
SPONTANEOUS: Occurs in absence of known mutagen
INDUCED: occurs in presence of known mutagen
Classifying mutations..
CELL TYPE:
SOMATIC VS GERMLINE
SOMATIC - occurs in non-reproductive cells
GERMLINE: occurs in reproductive cells
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
Classifying Mutations…
EFFECT ON FUNCTION =4
- LOSS OF FUNCTION (KNOCKOUT, NULL) - eliminates normal function
- HYPOMORPHIC (LEAKY) - reduces normal function
- HYPERMORPHIC - increases normal function
- GAIN OF FUNCTION (ECTOPIC EXPRESSION): expressed ar incorrect time in or in appropriate cell types.
Classifying Mutations…
MOLECULAR CHANGE = 5
- NUCLEOTIDE SUBSTITUTION = one base pair in duplex DNA replaced with a different base pair
- TRANSITION = pyrimidine (T OR C) to pyrimidine, or purine ( A or G) to purine
- TRANSVERSION: pyrimidine (T or C) to purine or purine (A or g) TO PYRIMIDINE
- INSERTION: One or more extra nucleotides present
- DELETION: One or more missing nucleotides
Classifying Mutations… EFFECT ON TRANSLATION = 4
- SYNONYMOUS (SILENT) = no change in amino acid encoded
- MISSENSE (NONSYNONYMOUS) = chnage in amino acid encoded
- NONSENSE (TERMINATION) = Creates translational termination codon (UAA, UAG or UGA)
- FRAMESHIFT = Shifts triplet reading of codons out of correct phase
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
Effects of mutations: DIAGRAM
LOOK AND UNDERSTAND DIAGRAM IN SLIDE 33
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
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
- 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)
“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)
How do mutations cause disease?
IMPORTANT TO LEARN THE DIAGRAM = SLIDE 36
