Chapter 11 + 13 (M2) Flashcards
Point mutation
substitution, insertion, or deletion of a single base pair in a gene
- occur during DNA replication
- rare per cycle, common across large populations
Measuring mutation rate (2 ways)
- Phenotypic level
10^-6 to 10^-8 / individual - DNA sequence level
10^-9 /base/replication
less at the sequence level bc studied in a controlled lab
Delbruck two mutation hypotheses
- mutations are random
- mutations arise from environmental triggers
Experiment showing mutations are random
Cultivated bacteria and infected them with a virus (T1 phage) at a specific generation and tested their resistance to virus
Resistant bacteria developed in a way consistent with random
mutation hypothesis
number of phage-resistant cells fluctuates substantially among populations as a result of random timing of mutation
Germ-line mutations
Mutations generated within gametes, can be
passed on to next generation
Somatic mutations
Typically generated during mitosis. Not passed on,
but could affect individual
- E.g. cancerous tumours
Point mutations: transition vs. transversion mutations
Transition
- purine to purine
- pyr to pyr
(A to G / T to C)
Transversion
- pyr to pur
- pur to pyr
(A to T or C)
Transition or transversion more common
Transition
flexible bp / wobble hypothesis with redundancy in genetic code
5 types of point mutations
- Synonymous (same aa)
- Missense (diff aa)
- Nonsense (early stop)
- Insertion
- Deletion
insertion and deletion MAY result in frameshift
MAY not if in an insertion that’s a multiple of 3
Forward mutation
wild to mutant allele
Reverse mutation (aka reversion)
3 types
mutant to wild-type allele
- True reversion
- mutation restores exact wild-type amino acid - Intragenic reversion
- mutation elsewhere in the same gene restores gene function - Second-site reversion
- mutation in a different gene restores wild phenotype
aka suppressor mutation
Blue flower example of second-site reversion
- two genes encode pigment transport protein
- mutation in one gene leads to reduced pigment
- mutation in second gene causes upregulates
second transport protein, restoring wild-type phenotype
Causes of point mutation (3)
- Mispaired nucleotides during replication
- Spontaneous nucleotide base change
- Mutagens (chemical or radiation)
Incorporating error
During …
example of mispaired nucleotides during replication
non-complementary base pairing (G:T / C:A)
without repair, replication leads to mutation
Depurination
example of a spontaneous nucleotide base change
The loss of a purine → apurinic site
If not repaired, DNA polymerase will put an adenine during replication
__ → A
- Common way for a G→A substitution to occur
Deamination
example of a spontaneous nucleotide base change
The loss of an amino
group (NH2) from a nucleotide base
- Methylated cytosine can undergo
deamination to become a thymine - This can lead to a mismatch, which
when repaired can cause a C-G pair to
become T-A
Classification of chemical mutagens (6)
- Nucleotide base analogs: a chemical with a similar structure to DNA.
Incorporates into DNA during replication and induce point mutation - Deaminating agents: removes amino groups. Can stimulate a C-G pair to become T-A
- Alkylating agents: add methyl or ethyl groups to nucleotide bases, causing a distortion in DNA helix, leading to mutations
- Oxidizing agents: oxidizes nucleotide base, usually resulting in a transversion
mutation - Hydroxylating agents: Add hydroxyl groups to a nucleotide base, usually resulting in a modified cytosine pairing with A
- Intercalating agents: molecules that fit between DNA base pairs, distorting
the DNA duplex, leading to lesions that may result in frameshift mutations
Mutagen
Anything that causes a mutation (a change in the DNA of a cell)
Ames test
A test to verify if a chemical is a mutagen
- Process involves exposing bacteria to a chemical in the presence
of enzymes extracted from a mammalian liver - Uses bacteria with mutations in several
genes that prevent histidine synthesis - Cultivates bacterial mutants on media
without histidine and test chemical - If bacterial mutants grow, then mutations
occurred in either mutated gene, allowing
bacteria to synthesize histidine - That result indicates the chemical has
mutagenic properties
How to test how mutagenic a chemical is
Counting colonies of bacteria in test
plates compared to control plates
evaluates how mutagenic a chemical is
UV radiation - how it causes mutations
- Thymine dimers can form from excessive
UV radiation exposure - These are covalent bonds between C5-C6
or C4-C6 of adjacent thymines - DNA repair mechanisms can repair these
dimers - However, if not repaired, can disrupt DNA
replication, inducing mutations in the
process - Primary cause for the strong association
between excessive UV exposure and skin
cancer
Is all high-radiation mutagenic?
where do they cause mutations (which line?)
Yes!
UV, x-rays, gamma rays, cosmic rays
Radiation exposure induces mutations
in germ-line, which may get passed on
to offspring
Base excision repair
Removal of an incorrect or damaged DNA base and repair by synthesis of a new strand segment (nick translation)
Nick translation
DNA polymerase initiates removal and replacement of nucleotides and DNA ligase seals the sugar-phosphate backbone
Process of base excision repair
- DNA N-glycosylate recognizes a base-pair mispatch
- Removes incorrect = creates an apyrimidinic site
- AP endonuclease generates a single-stranded nick on the 5’ side
- DNA poly removes and replaces several nucleotides of the nicked strand
Nucleotide excision repair
Removal of a strand segment containing
DNA damage and replacement by new DNA synthesis
Often used to repair UV-induced damage to DNA (aka UV repair)
Process of nucleotide excision repair
similar to BER
- Enzymes recognize and bind to damaged region
- Segment of nucleotides is removed from damaged strand
- DNA polymerase fills the gap
- DNA ligase seals the sugar-phosphate backbone
Mismatch repair
Removal of a DNA base-pair mismatch by
excision of a segment of the newly synthesized strand followed by resynthesis of the excised segment
Process of mismatch repair
- During DNA replication, parental strand is usually
methylated, while daughter strand is not - MutH protein binds to unmethylated daughter strand
- MutS binds to basepair mismatch
- MutL connects MutH to MutS
- MutH cleaves daughter strand
- DNA polymerase synthesizes gap
Translesion DNA Synthesis
Error-prone
- Unrepaired DNA damage can block DNA polymerase III, causing it to stall
causes SOS response
SOS response
Repair system in E. coli used in response to massive
DNA damage that blocks DNA polymerase III
- Activates translesion DNA polymerases (pol V) that bypass
these lesions and synthesizes short DNA segments - This specialized polymerase has no proof-reading abilities and therefore has a high mutation rate
Double-stranded break repair
- Double stranded breaks (DSBs) lack a template for DNA repair
- Can cause chromosome instability, cell death, and cancer
Double-stranded break repair –> two mechanisms
- Nonhomologous end joining (NHEJ)
- Synthesis-dependent strand annealing (SDSA)
Nonhomologous end joining (NHEJ)
Error-prone and can lead to mutation
- When double stranded breaks occur, both
strands of DNA are trimmed into even
“blunt ends” and then rejoined with DNA
ligase - Trimming leads to loss of nucleotides that
cannot be replaced. - May lead to frameshift mutations
NHEJ in-depth
- DSB from x-ray or oxidative damage
- Ku80-Ku70-PKcs protein complex binds DNA ends
- Ends trimmed = loss of nucleotides
- DNA ligase reforms duplex (ligates blunt ends)
Synthesis-dependent strand annealing (SDSA)
Error-free process
- After DNA replication, if one chromatid gets damaged on both
DNA strands, the intact sister chromatid can help repair - Strand invasion offers a template to synthesize new DNA
- Similar process to homologous
recombination but repairs DNA
SDSA in-depth
- DSB from x-ray or oxidative damage
- Nucleases digest park of broken strand and Rad51 binds to undamaged
- Sister chromatid invades strand
= creates a D loop with a replication fork - New strand synthesis using intact strand as template
- Partial strand excision; duplexes reform; strands ligated
CRISPR
Inject an embryo with a plasmid or mRNA to express:
* Cas9 nuclease enzyme identifies and cuts target
* Guide RNA (same sequence as defective) to guide Cas9 to genomic target
Knock-out
- nuclease-induced double-stranded break
- NHEJ to delete gene of interest in the absence of a template
Knock-in
- donor template added in like SDSA style
- re-insertion of modified gene
Transposable Genetic Elements (TGE)
- what are they
“selfish DNA elements”
- DNA sequences that move within the genome through transposition
(facilitated by transposase enzyme) - Different TGEs vary in length, sequence composition, and copy
number
TGE - appearance
- Terminal inverted repeats on its ends (part of TGE)
- The inserted TGE is bracketed by flanking direct repeats (not part of TGE)
TGE - two types of movement
- Non replicative transposition: excision of the element from its original
location and insertion in a new location (cut & paste) - Replicative transposition: duplication of the element and insertion of the copy in a new location (copy & paste)
Transposition in-depth
- Staggered cuts cleave DNA of target sequence
- Result in single stranded ends (hanging over)
- The transposable element is inserted into the target sequence
- Gaps are filled by DNA polymerase
2 types of transposons
- DNA transposons: transpose as DNA sequences
* Replicative: copy & paste
* Non-replicative: cut & paste - Retrotransposons: are composed of DNA, but transpose
through an RNA intermediate
* DNA → RNA → reverse transcribed into DNA
* use enzyme reverse transcriptase
How do transposons cause mutations
They insert themselves
into crucial genetic regions (e.g. coding region, promoter, etc)
ex.
- hemophilia A, Coffin-Lowry syndrome
- round vs wrinkled peas
Transposition in Drosophila melanogaster
TGEs not found in wild-captured flies prior to 1960!
- Suggests around 1960, TGEs, called P-elements, were introduced into flies and they proliferated fast
P-elements (pre-CRISPR)
transposable elements
found in Drosophila
Utilized in a technique to generate transgenic flies
P-element process
- Clone gene of interest into plasmid flanked
by inverted repeats characteristic of TGE - Inject embryo with plasmid and transposase
enzyme - Gene of interest will randomly insert itself
into genome of embryo
Epigenetics
the study of genes above inheritance
Genetics
the study of inheritance
Gene
to produce (context of reproduction)
2 examples of epigenetics
Agouti mice
Dutch post famine
Five Features of Epigenetic Modifications
- Epigenetic modification patterns alter chromatin structure
- They are transmissible during cell division
- They are reversible
- They are directly associated with gene transcription
- They do not alter DNA sequence
Euchromatin
Loosely compacted genomic regions (chromatin), more
transcriptionally active
Heterochromatin
Densely compacted chromatin, less transcriptionally active
Constitutive heterochromatin
genomic regions that are always heterochromatin
Facultative heterochromatin
genomic regions that switch back and forth between euchromatin
and heterochromatin
Position effects
- characteristics of a region
may be transcriptional hotspots or transcriptional coldspots
Nucleosome
Structure consisting of DNA wound around 8
histone proteins
* 2 H2A-H2B dimers + 1 H3-H4 tetramer
- Histone proteins enable DNA to coil around it
Acetylation (chromatin modification)
Relaxes histone/DNA interaction by neutralizing positively
charged histones
Histone acetyltransferases (HATs)
add acetyl groups to histones, leading to euchromatin
Histone deacetylases (HDACs)
remove acetyl groups from histones,
leading to heterochromatin
Methylation
typically associated with heterochromatin, however methylation can also lead to
euchromatin
can lead to either hetero or eu
Histone methyltransferases (HMTs)
add methyl groups to histones
Histone demethylases (HDMTs)
remove methyl groups from histones
Position effect variegation (PEV)
occurs when
heterochromatic areas spread into euchromatin, silencing transcription of genes
PEV in Drosophila
Mutant line where an
inversion on the X chromosome placed the white gene near the centromere in a heterochromatic region
- Wild-type allele for the white gene produces red eyes; gene named after mutant phenotype
- That created a mosaic where some cells in the fly compound eye were wild-type and other mutated
- Yet these flies are genotypically wild-type!
genotypically red (active w+ allele)
phenotypically mosaic (w+ silenced by heterochromatin = white)
E(var) mutations
- Short for “enhancers of position effect variegation”
- Enhance mutant
phenotypes by encouraging spread of heterochromatin
mostly white
Sur(var) mutations
- Short for “suppressors of position effect variegation”
- Restrict heterochromatin
spread, encouraging wild-type phenotype
mostly red
X-inactivation in female placental mammals
Occurs early in embryonic development
- Any given cell inactivates either the
maternally inherited X chromosome or the
paternally inherited X chromosome on a
random basis - All cells in a female’s body are
mosaics of two cell types: one expresses
the maternal X chromosome, the other
expresses the paternal X chromosome
Also considered an epigenetic
phenomenon
Colour-blind mosaicism
both seen for women carrying colourblind allele
50-50 x-inactivation
- half of cells work
- normal vision
Skewed X-inactivation
- most cells don’t work
- red-green colour blind
Long noncoding RNAs (lnc RNA)
- Long RNA that lack open reading frames
- Play a role in gene regulation in eukaryotic cells
- Studied in stem cells of mice embryos
- Thought to act as scaffolds that link to regulatory proteins, affecting
chromatin structure - Involved in X-inactivation
ex. X-inactivation-specific transcript (Xist)
Xist gene vs. Xist RNA
Xist gene is in the X-inactivation center (XIC) on the X chromosome
* Active in heterochromatic X
chromosome
* Inactive in euchromatic X chromosome
Xist RNA produced on X chromosome
to be inactivated
* It spreads along the length of the
chromosome and inactivates almost
all the genes, silencing the chromosome
Genomic imprinting
Heritable epigenetic phenomenon
- Involves some genes whose expression in offspring depends on
the parent that passed it on
Can be X-linked or autosomal
Maternal imprinting
Allele passed on by mother inactivated; therefore offspring express allele from father
only females switch alleles off when passing them on
only affected males or carrier males can have affected children
Paternal imprinting
Allele passed on by father inactivated; therefore offspring express allele from mother
only males switch alleles off when passing them on
only affected females or
carrier females can have affected
children
IGF2 and H19
Both genes close to each other on
chromosome 11
- H19 is only expressed on maternally
inherited chromosome
(enhancer drives, insulator blocks IGF2) - IGF2 (insulin growth factor 2) is only expressed on paternally inherited chromosome
(methylation inactivates the insulator ICR, blocks H19 expression, drives IGF2 expression)
Russell-Silver syndrome
both chromos have maternal expression pattern
= underweight infants
Beckwith-Wiedemann syndrome
both chromos have paternal expression pattern
= overgrowth of tissue
Agouti mice epigenetics
- a modified agouti gene leads to yellow coat colour and extreme obesity
- female mice fed a certain diet during gestation lead to wild-type offspring, despite inheriting the modified agouti gene
- The modified diet was rich in methyl factors. This lead to increased methylation
and silencing of the modified agouti gene.
Dutch post-famine epigenetics
- those that survive famine during WWII had increased risk of heart disease, diabetes, and obesity
- compared to those that did not live through famine
- IGF2 gene less methylated in citizens born during the famine.
- Siblings in the same families born after the famine have higher methylation of
IGF2
