Molecular Basis of Mutation Flashcards
(122 cards)
1
Q
what is a mutation?
A
an alteration in the nucleotide sequence of a DNA molecule
2
Q
what are the 2 ways mutation can occur?
A
- errors in DNA replication (spontaneous mutations)
- caused by mutagens
3
Q
why don’t mutations caused by errors in DNA replication happen often?
A
DNA polymerases proofreading properties
4
Q
can mutations be caused by mistakes made by DNA polymerase?
A
yes
5
Q
what’s base tauterism?
A
mutations caused by isomers of bases with slightly different chemical structures
6
Q
how does base tauterism affect DNA?
A
affect the hydrogen bonds between base pairs, leading to incorrect bonding e.g. A-C
7
Q
do amino- bases show normal base pairing?
A
yes
8
Q
do keto- bases show normal base pairing?
A
yes
9
Q
do imino- bases show normal base pairing?
A
no
10
Q
do enol- bases show normal base pairing
A
no
11
Q
what are mutagens?
A
chemical or environmental agents that cause changes in DNA molecules
12
Q
what are the 2 important ways in which mutagens can cause mutations?
A
- base analogs
- direct structural change
13
Q
what’s a base analog?
A
a chemical that can substitute for a normal nucleobase in nucleic acids
14
Q
what adds base analogs?
A
polymerase
15
Q
what are direct structural changes?
A
when a physical component is added which changes the DNA
16
Q
which is an important base analog?
A
5-bromouracil (5bU)
17
Q
what is 5bU an analog of?
A
Thymine
18
Q
what does keto-5bU pair with?
A
adenine
19
Q
what’s the problem with 5bU?
A
the enol tautomer is very common and it pairs with G instead of T
20
Q
what do deaminating agents do?
A
change the structures of some nucleotides by removing an Amine group
21
Q
give 2 examples of deaminating agents
A
- nitrous acid
- sodium bisulphite
22
Q
what does deamination of adenine give?
A
hypoxanthine
23
Q
what does hypoxanthine base pair with?
A
C (not T)
24
Q
what does the deamination of cytosine give?
A
uracil
25
what does uracil base pair with? instead of?
A instead of G
26
what does the deamination of Guanine give?
xanthine
27
what does xanthine do?
blocks DNA replication
28
what are 2 other examples of agents causing structural changes to nucleotides?
- alkylating agents (add alkyl groups)
| - intercalating agents (insert between pase pairs)
29
give an example of an aklylating agent
ethylmethane sulphonate
30
give an example of an intercalating agent
ethidium bromide
31
what does ethidium bromide do?
causes errors during DNA replication e.g. polymerase adding in an additional base
32
what can Ultraviolet radiation do?
cause base dimerization
33
what is base dimerization?
where bases on the same strand join together
34
which dimers are particularly bad?
Thymine dimers
35
what can heat do?(2)
cause detachment of bases
| can give rise to AP site
36
what's an AP site?
apurine/apyrimidinic - leads to missing BP through the hydrolysis of B-N- glycosidic bond
37
what are the 4 types of mutation repair?
- direct
- excision
- mismatch
- nonhomologous end joining
38
what's direct repair?
damaged nucleotide--> enzyme comes in --> changes it --> returns to correct nucleotide (rare)
39
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
40
what's mismatch repair?
the same as excision, except the damage is mismatched nucleotides base pairing
41
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
42
what sort of nucleotide alteration does direct repair correct?
alternation caused by mutagen
43
how common is direct repair?
quite uncommon
44
for each change in a nucleotide, what is needed in direct repair?
a specific enzyme
45
how many enzymes do humans have involved in direct repair?
12
46
what are the 2 types of excision repair?
base
| nucleotide
47
what happens in base excision repair?
only the single, mutated base is removed and replaced
48
what happens in nucleotide excision repair?
a longer piece of DNA containing the altered bases is removed
49
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
50
is nucleotide excision repair common?
yes
51
what carries out nucleotide excision repair?
UvrABC endonuclease
52
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
53
what sort of errors does mismatch repair correct?
errors in replication
54
how is the parent or daughter strand of DNA distinguished to find which contains the error?
the parent strand is methylated - in e.coli
55
what recognises mismatch?
MutH and MutS enzymes
56
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
```
57
what's the difference between endonucleases and exonucleases?
endonucleases go from the end
| exonucleases go from the middle
58
what does Nonhomologous end joining correct?
DNA breaks (complete cuts of both strands)
59
in nonhomologous end joining what distinction is important?
the real breaks in DNA from the ends of chromosomes
60
what distinguishes the real breaks from the ends of chromosomes?
telomeres mark the natural ends of chromosomes
61
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
62
what's a point mutation?
when one base pair is replaced with another
63
what's a transition point mutation?
purine to purine/ pyrimidine to pyrimidine (same)
64
what's a transversion point mutation?
purine pyrimidine (different)
65
what's an insertion mutation?
where one or more base pairs are inserted
66
what's a deletion mutation?
one or more base pairs are deleted
67
what's an inversion mutation?
2+ base pairs are excised and reinserted in the opposite direction
68
what's a synonymous mutation?
where the point mutation has no effect on the amino acid sequence
69
what's a synonymous mutation AKA?
silent mutation
70
what's a non-synonymous mutation?
where a point mutation changes the AA sequence
71
what's a non-synonymous mutation AKA?
missense mutation
72
what's a nonsense mutation?
where a point mutation changes the AA into a stop codon
73
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)
74
what's a readthrough mutation?
where a point mutation changes a stop codon into an AA (causes longer proteins)
75
what's a frameshift mutation?
a change in the reading frame, all codons downstream of the mutation are changed
76
how could an insertion/deletion not change the reading frame?
if a whole codon (3 bases) are inserted/deleted
77
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
78
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
79
what's the most common CFTR mutation in the UK?
F508 mutation
80
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
81
what's the second most common CFTR mutation?
G542X mutation
82
what occurs in the G542X mutation?
nonsense mutation changes glycine to stop codon
| CFTR protein isn't made
83
what's the 3rd most common CFTR mutation?
G551D
84
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
85
what's a second site reversion?
another mutation restores the correct AA sequence
86
what's a supression?
a mutation in the tRNA genes supresses nonsense mutations in protein coding genes
87
what is a phenotype?
biological characteristics
88
what are most organisms, in terms of gene number?
diploid
89
if there's a mutation in one gene, does it always affect the genotype?
no- it could be recessive
90
what's haplosufficiency?
when a mutation in only one gene is all that's required to cause disease
91
give an example of haplosufficiency
Alagille syndrome
92
what's trisomy?
3 copies of a chromosome
93
what causes down syndrome?
trisomy of chromosome 21
94
what is monosomy also known as?
aneuploidy
95
what does monosomy 7 lead to?
bone marrow failure --> high risk of leukemia
96
what is chromosome translocation?
when part of one chromosome becomes attached to another chromosome
97
what's the Philadelphia chromosome a common cause of?
leukemia
98
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
99
what is molecular cloning?
cutting, joining and propagating recombinant DNA
100
very generally, how does molecular cloning occur?
- isolate DNA
- cut DNA
- insert into vector (recombinant)
- introduce recombinant vector into bacteria
- amplify recombinant growth
101
what cuts/splices DNA?
restriction endonucleases
102
what sort of sequence do restriction endonucleases cut?
palindromic sequences
103
what do different restriction endonucleases have?
different sequence specificities
104
what's a palindromic sequence?
one where the complementary sequence is the same
105
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
106
what 2 enzymes allow the cut DNA to join with bacterial vector?
ligase
| photophatase
107
what does phosphatase do?
allows a stable ligated molecule of recombinant plasmid
108
what are DNA libraries?
isolation and separation of individual sequences within a cell
109
what genes are found in a genomic library?
all (genome)
110
what genes are found in the transcriptome/ DNA library?
only expressed genes
111
what's cDNA (complementary DNA)?
- makes up the transcriptome
- libraries from different tissues make up different sequences
- no unsubscribed sequences
112
what's genomic DNA?
- all sequences in genome
- same sequences in all tissues
- introns and exons
113
what is the process of mRNA--> cDNA?
reverse transcription
114
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
115
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
116
is enhancer found in cDNA or genomic?
genomic
117
is promoter found in cDNA or genomic?
genomic
118
is 5'UTR found in cDNA or genomic?
both
119
are exons found in cDNA or genomic?
both
120
is 3'UTR found in cDNA or genomic?
both
121
is intergenic DNA found in cDNA or genomic?
genomic
122
are introns found in cDNA or genomic?
genomic
