Molecular Basis of Mutation Flashcards
what is a mutation?
an alteration in the nucleotide sequence of a DNA molecule
what are the 2 ways mutation can occur?
- errors in DNA replication (spontaneous mutations)
- caused by mutagens
why don’t mutations caused by errors in DNA replication happen often?
DNA polymerases proofreading properties
can mutations be caused by mistakes made by DNA polymerase?
yes
what’s base tauterism?
mutations caused by isomers of bases with slightly different chemical structures
how does base tauterism affect DNA?
affect the hydrogen bonds between base pairs, leading to incorrect bonding e.g. A-C
do amino- bases show normal base pairing?
yes
do keto- bases show normal base pairing?
yes
do imino- bases show normal base pairing?
no
do enol- bases show normal base pairing
no
what are mutagens?
chemical or environmental agents that cause changes in DNA molecules
what are the 2 important ways in which mutagens can cause mutations?
- base analogs
- direct structural change
what’s a base analog?
a chemical that can substitute for a normal nucleobase in nucleic acids
what adds base analogs?
polymerase
what are direct structural changes?
when a physical component is added which changes the DNA
which is an important base analog?
5-bromouracil (5bU)
what is 5bU an analog of?
Thymine
what does keto-5bU pair with?
adenine
what’s the problem with 5bU?
the enol tautomer is very common and it pairs with G instead of T
what do deaminating agents do?
change the structures of some nucleotides by removing an Amine group
give 2 examples of deaminating agents
- nitrous acid
- sodium bisulphite
what does deamination of adenine give?
hypoxanthine
what does hypoxanthine base pair with?
C (not T)
what does the deamination of cytosine give?
uracil
what does uracil base pair with? instead of?
A instead of G
what does the deamination of Guanine give?
xanthine
what does xanthine do?
blocks DNA replication
what are 2 other examples of agents causing structural changes to nucleotides?
- alkylating agents (add alkyl groups)
- intercalating agents (insert between pase pairs)
give an example of an aklylating agent
ethylmethane sulphonate
give an example of an intercalating agent
ethidium bromide
what does ethidium bromide do?
causes errors during DNA replication e.g. polymerase adding in an additional base
what can Ultraviolet radiation do?
cause base dimerization
what is base dimerization?
where bases on the same strand join together
which dimers are particularly bad?
Thymine dimers
what can heat do?(2)
cause detachment of bases
can give rise to AP site
what’s an AP site?
apurine/apyrimidinic - leads to missing BP through the hydrolysis of B-N- glycosidic bond
what are the 4 types of mutation repair?
- direct
- excision
- mismatch
- nonhomologous end joining
what’s direct repair?
damaged nucleotide–> enzyme comes in –> changes it –> returns to correct nucleotide (rare)
what’s excision repair?
region around the damaged nucleotide is removed and then the region is resynthesized, damage on one but but template on other used for resynthesis
what’s mismatch repair?
the same as excision, except the damage is mismatched nucleotides base pairing
what’s nonhomologous and joining repair?
when a clear cut/clean break is in DNA e.g. by radiation or chemical. they are brought back together to give a correct sequence/ intact DNA
what sort of nucleotide alteration does direct repair correct?
alternation caused by mutagen
how common is direct repair?
quite uncommon
for each change in a nucleotide, what is needed in direct repair?
a specific enzyme
how many enzymes do humans have involved in direct repair?
12
what are the 2 types of excision repair?
base
nucleotide
what happens in base excision repair?
only the single, mutated base is removed and replaced
what happens in nucleotide excision repair?
a longer piece of DNA containing the altered bases is removed
what’s the mechanism of base- excision repair in E.coli?
- begins with the removal of the damaged base by a DNA- glycosylase enzyme (breaks glycosidic bond)
- resulting AP site is then filled in (enzymes remove sugar and replice the entire nucleotide- not just the base), gap filled in and phosphodiester bond is formed
is nucleotide excision repair common?
yes
what carries out nucleotide excision repair?
UvrABC endonuclease
what’s the mechanism of nucleotide excision repair?
- the damaged nucleotide causes helix distortion
- this is detected by the endonuclease (A detects distortion, B binds to the damaged area), the endonuclease is now active
- B subunit cuts downstream of damage and C subunit cuts upstream of damage
- Helicase II removes this section
- B subunit remains in the gap, protecting exposed DNA
- new strand is synthesised and is ligated together
what sort of errors does mismatch repair correct?
errors in replication
how is the parent or daughter strand of DNA distinguished to find which contains the error?
the parent strand is methylated - in e.coli
what recognises mismatch?
MutH and MutS enzymes
what’s the mechanism of mismatch repair?
MutS recognises the mismatched base and MutH binds upstream of the damage MutH cleaves this section DNA helicase II removes this strands strand now exposed strand repaired
what’s the difference between endonucleases and exonucleases?
endonucleases go from the end
exonucleases go from the middle
what does Nonhomologous end joining correct?
DNA breaks (complete cuts of both strands)
in nonhomologous end joining what distinction is important?
the real breaks in DNA from the ends of chromosomes
what distinguishes the real breaks from the ends of chromosomes?
telomeres mark the natural ends of chromosomes
what’s the mechanism of nonhomologous end joining in humans?
- double strand break
- Ku proteins attach to both sides of break
- they’re attracted to both ends of broken DNA and eachother
- pull DNA back together
what’s a point mutation?
when one base pair is replaced with another
what’s a transition point mutation?
purine to purine/ pyrimidine to pyrimidine (same)
what’s a transversion point mutation?
purine pyrimidine (different)
what’s an insertion mutation?
where one or more base pairs are inserted
what’s a deletion mutation?
one or more base pairs are deleted
what’s an inversion mutation?
2+ base pairs are excised and reinserted in the opposite direction
what’s a synonymous mutation?
where the point mutation has no effect on the amino acid sequence
what’s a synonymous mutation AKA?
silent mutation
what’s a non-synonymous mutation?
where a point mutation changes the AA sequence
what’s a non-synonymous mutation AKA?
missense mutation
what’s a nonsense mutation?
where a point mutation changes the AA into a stop codon
what does the effect on a nonsense mutation depend on?
how far into the gene coding for a protein the stop codon occurs (worse if it’s found nearer the beginning)
what’s a readthrough mutation?
where a point mutation changes a stop codon into an AA (causes longer proteins)
what’s a frameshift mutation?
a change in the reading frame, all codons downstream of the mutation are changed
how could an insertion/deletion not change the reading frame?
if a whole codon (3 bases) are inserted/deleted
what’s the CFTR protein?
a chloride channel on the cell surface and is responsible for proper balance of salt and water within a cell
what does a mutation in CFTR cause?
dysfunction in the salt and water balance leading to thick mucous and excessive loss of salt in sweat
what’s the most common CFTR mutation in the UK?
F508 mutation
what occurs in the F508 mutation?
deleltion of 3 nucleotides, removes a codon for phenylalanine
CFTR protein still made but doesn’t reach cell surface
what’s the second most common CFTR mutation?
G542X mutation
what occurs in the G542X mutation?
nonsense mutation changes glycine to stop codon
CFTR protein isn’t made
what’s the 3rd most common CFTR mutation?
G551D
what occurs in the G551D mutation?
a non- synonymous mutation changes a glycine (G) to an aspartic acid (D)
CFTR protein is made and reaches cell surface, however works at 4% the normal rate
what’s a second site reversion?
another mutation restores the correct AA sequence
what’s a supression?
a mutation in the tRNA genes supresses nonsense mutations in protein coding genes
what is a phenotype?
biological characteristics
what are most organisms, in terms of gene number?
diploid
if there’s a mutation in one gene, does it always affect the genotype?
no- it could be recessive
what’s haplosufficiency?
when a mutation in only one gene is all that’s required to cause disease
give an example of haplosufficiency
Alagille syndrome
what’s trisomy?
3 copies of a chromosome
what causes down syndrome?
trisomy of chromosome 21
what is monosomy also known as?
aneuploidy
what does monosomy 7 lead to?
bone marrow failure –> high risk of leukemia
what is chromosome translocation?
when part of one chromosome becomes attached to another chromosome
what’s the Philadelphia chromosome a common cause of?
leukemia
what happens in a philadelphia chromosome?
the control region of the gene for cell division is lost, therefore the gene is switched on all the time and cell division is uncontrolled
what is molecular cloning?
cutting, joining and propagating recombinant DNA
very generally, how does molecular cloning occur?
- isolate DNA
- cut DNA
- insert into vector (recombinant)
- introduce recombinant vector into bacteria
- amplify recombinant growth
what cuts/splices DNA?
restriction endonucleases
what sort of sequence do restriction endonucleases cut?
palindromic sequences
what do different restriction endonucleases have?
different sequence specificities
what’s a palindromic sequence?
one where the complementary sequence is the same
How is the DNA cut?
by restriction endonucleases
cut is staggered- cut at the same nucleotides on both sides but not parallel
this means that there’s an overhang- sticky end
what 2 enzymes allow the cut DNA to join with bacterial vector?
ligase
photophatase
what does phosphatase do?
allows a stable ligated molecule of recombinant plasmid
what are DNA libraries?
isolation and separation of individual sequences within a cell
what genes are found in a genomic library?
all (genome)
what genes are found in the transcriptome/ DNA library?
only expressed genes
what’s cDNA (complementary DNA)?
- makes up the transcriptome
- libraries from different tissues make up different sequences
- no unsubscribed sequences
what’s genomic DNA?
- all sequences in genome
- same sequences in all tissues
- introns and exons
what is the process of mRNA–> cDNA?
reverse transcription
how is a cDNA library made?
- isolate mRNA
- convert to cDNA (reverse transcriptase)
- insert into a vector and transform (into bacteria)
- colonies of bacteria grow- 1 sequence per colony
how is a Genome DNA library made?
isolate DNA
cut it up- restriction endonucleases
insert into vector
grow colonies- each colony has a different fragment of DNA to represent your genome
is enhancer found in cDNA or genomic?
genomic
is promoter found in cDNA or genomic?
genomic
is 5’UTR found in cDNA or genomic?
both
are exons found in cDNA or genomic?
both
is 3’UTR found in cDNA or genomic?
both
is intergenic DNA found in cDNA or genomic?
genomic
are introns found in cDNA or genomic?
genomic
