Download GENE MUTATION = POINT MUTATION at the DNA level: at the level

MUTATION JARGON
GENE MUTATION = POINT MUTATION
(scale of mutation is small and is localized to a specific region,
a single nucleotide or a few adjacent base pairs)
↓
at the DNA level:
Ë single base pair substitutions: transitions & transversions
Ë single (or a few) base pair addition or deletion: indels
Ë gene mutation by transposon insertion
at the level of
gene expression:
at the protein
level:
promoter mutations
splicing mutations
regulatory mutations
nonsense
missense
[neutral]
silent
frameshift
at the level of gene function:
loss-of-function
gain-of-function
[neutral]
1
CHROMOSOME MUTATION
• involves segments of chromosomes or
whole chromosomes or whole genomes
• alterations in chromosome structure
and number
• deletion, duplications, translocations
and inversions
• CNVs: copy number variations
Mutation: any heritable change in the genetic material (excludes
changes caused by normal recombination events)
S--t happens
The integrity of genomic DNA is constantly under threat, even in
perfectly healthy cells. DNA damage can result from the action of
endogenous reactive oxygen species, or from stochastic errors in
replication or recombination, as well as from environmental and
therapeutic genotoxins.
2
Spontaneous mutations: a mutation that occurs in the absence
of known mutagens
• uncorrected errors* that occur during DNA replication, repair
or recombination
• spontaneous lesions that occur to the DNA molecule under
normal physiological conditions and that are not repaired by the
cell’s DNA excision repair processes -- see text for examples of
excision repair process
* what quality control processes are in place?
3
Here’s what happens if an error slips by the quality control processes:
After two rounds of replication, the mutation is present in both strands
4
Transition mutation: a specific type of mutation that
involves the replacement of a purine with a purine or a
pyrimidine with a pyrimidine:
for example a GCèAT change
______ G____
_____ A____
______ C ____ è_____ T____
5
Spontaneous mutations: a mutation that occurs in the absence
of known mutagens
• uncorrected errors that occur during DNA replication, repair or
recombination
• spontaneous lesions that occur to the DNA molecule under
normal physiological conditions and that are not repaired by the
cell’s DNA excision repair processes -- see text for examples of
excision repair process
6
Your DNA is under constant assault:
Science Dec. 23, 1994
• Every second that you read this, the DNA in each cell of your
body is being damaged
• Chemical bonds are breaking
• DNA strands are snapping
• Nucleotide bases are flying off
• Each cell loses more than 10,000 bases per day just from
spontaneous breakdown of DNA at body temperature
• Meanwhile many cells are dividing and therefore copying DNA
and each copy introduces the possibility of error
• Exposure to carcinogens adds to the injury and causes strange
new forms to sprout from the double helix
7
(After T. Lindahl, Nature 362:709–715, 1993)
The sites on each nucleotide that are known to be modified by
• spontaneous oxidative damage (red arrows)
• hydrolytic attack (blue arrows)
• uncontrolled (non-enzymatic) methylation by the methyl group donor Sadenosylmethionine (green arrows)
• WIDTH of each arrow indicates the relative frequency of each event
8
Spontaneous loss of guanine results in an abasic site
Depurination is a very common
spontaneous lesion
It is estimated that a single
mammalian cell loses 10,000
purines from its DNA in a 20 hour
period (see below)
Rate of depurination: 4 X 10-9 sec-1 at pH7.4
It may then be predicted that an E. coli cell, growing
with a generation time of 40 min at 37degC, should lose
0.5 purine/chromosome in each generation. For a
mammalian cell, which contains ~800 times more DNA
than E. coli and grows with a generation time of 20 hr,
12,000 purines should be lost from the DNA in each cell
generation due to hydrolysis. However, as at least 50%
of the DNA is present as nucleohistone in this case and
therefore may be protected, it is possible that 2,00010,000 depurination events per generation would be a
better estimate. In the same fashion, a long-lived, nongrowing mammalian cell, e.g., a human nerve cell would
lose ~108 purines from its DNA during the lifetime of
the individual, or ~3% of its total amount of purine
residues in DNA: ARGGHH: REPAIR NEEDED
BIOCHEMISTRY, VOL. 11, NO. 19, 1972
9
Most AP sites are repaired by the base excision repair system, which
deals with small scale lesions that don’t distort the helix
Uracil DNA glycosylase flips a uracil residue out of the duplex, shown in yellow.
10
Unrepaired depurination of DNA results in abasic sites -- a common
spontaneous lesion
spontaneous loss of G from ribose
---------G-------------------C-----------
AP = abasic = apurinic/apyrimidinic
--------- (AP)--------------------C-----------
LESION REPAIRED
SEE figure 15-23 in 9th or 16-20 in
10th: Base excision repair
LESION NOT
REPAIRED and cell
entere S PHASE
3’ ----------(AP)--------------5’
5’----------|
<----G---------------- 5’
5’--------------C-----------------3’
11
12
So-called bypass polymerases can move past a lesion in the template
High fidelity replicases cannot
spontaneous loss of G from ribose
------G ----------------(AP)--------------C-----------------C--------repaired*
DNA replication
occurs: unrepaired
strand is template
for one of the
daughter strands
--------(AP)--------another round of
--------- A--------replication
**bypass polymerases will
insert a purine opposite an abasic site
---------G ----------------C---------
-----------T------------------ A---------
13
*SEE figure
16-20 : Base
excision repair
** SEE figure
16-24
Transversion mutation
a specific type of mutation that involves the replacement of a purine with
a pyrimidine or a pyrimidine with a purine:
for example a GCèTA change
______ G____
_____ T____
______ C ____ è _____A____
14
Addition or deletion of one or a few bases can occur
spontaneously: such a lesion typically results from
“strand slippage” during DNA replication
Recall triplet repeat
expansion and
contraction in
Fragile X
Also, file this info
away and retrieve
during our
discussion of DNA
fingerprinting
15
The DNA sequences shown above are more prone to strand
slippage than, say, the sequence shown below:
3’ CTAGACTCAGTGTA….5’ template strand
5’ GATCTGA…..3’ daughter strand
Why?
Intercalating agents induce mutations that involve the
addition or deletion of a small number of base pairs: see
pg 565-56 of 10th
16
mutagen: any agent that increases the mutation rate above
the background, or spontaneous, rate
induced mutation: mutation occurring in the presence of a
mutagen
read about this class of mutagens in your text (section 16.3)
base analog mutagen: chemically resemble one of the purine
or pyrimidine bases
17
18
• Benzo[a]pyrene is a compound
found in cigarette smoke
• Hydrocarbons like
benzopyrene are insoluble in
water
• In the liver and lungs such
compounds are chemically
modified to make them more
soluble so the body can excrete
them.
• Intermediates that are
generated include epoxides
that react with guanine
Skull with Burning Cigarette
Artist?
19
The soluble product of the
reactions includes
benzo(a)pyrene 7,8 diol9,10 epoxide, which can
attack the electron rich area
of a DNA base
The enzyme oxygenase
catalyzes steps 1 and 3
The lungs have a system
for inducing synthesis of
oxygenase when levels of
benzo(a)pyrene are high
20
Cigarette smoke carcinogens such as benzo[a]pyrene are implicated in
the development of lung cancer:
• about 60% of human lung cancers involve mutations in a tumor
suppressor gene called p53
• a mutation database exists that includes more than 500 entries of
sequenced p53 genes from lung cancer cells
• a large percentage of these 500 entries have GC to TA transversions
(see next page for mechanism)
• such mutations are hallmarks of mutagenesis involving polycyclic
aromatic hydrocarbons
21
What happens to the modified G*?
1. The defective base is hydrolyzed (either spontaneously or via a repair
process; this leaves a deoxyribose with no base attached -- an apurinic site
2. If the apurinic site is not repaired before the next round of DNA
replication, an A typically is inserted opposite the “empty site”
3. After another round of DNA replication, the GC base pair has been
converted to a TA base pair
4. See diagram next page
22
Unrepaired abasic sites
* = benzo(a)pyrene 7,8 diol-9,10 epoxide
a pack a day
------G *-------------C---------
#1
---------(AP)-----------------C---------
repaired
(AP)=abasic site
#2
---------(AP)----------------- A--------bypass polymerases will insert a
purine opposite an abasic site
#3
---------G ----------------C---------
#2 DNA replication
occurs: unrepaired
DNA strand is
template for one of
the daughter
strands
#3 another round
of replication
----------T------------------ A---------
23
24