DNA mutation and repair Flashcards
Any change made to the DNA sequence or chromosome structure
Mutation
what can mutation create
Can lead to disease/death
create new alleles (evolution)
are the changes of mutation permanent?
yes
how are mutations classified
1) size
2) what causes them
3) Type of cell that contains mutated DNA
Large segments of chromosomes are deleted, inverted, moved, or duplicated
Chromosomal mutations
Smaller changes in the DNA sequences
Gene mutations
Some due to natural biochemical events
spontaneous mutations
Others helped along by some artificial factor (chemicals, radiation, viral)
induced mutations
Arise in the DNA of somatic cells (normal diploid)
Somatic mutations
what type of mutation are NEVER passed onto the next generation
Somatic mutations
Mutations arise in the DNA of gamete-forming tissue (those cells that produce sperm and eggs)
Germ-line mutations
what mutations can be transmitted to offspring
Germ-line mutations
mutations incompatible with life
lethal mutations
mutations that lead to prenatal death
embyronic lethal mutations
mutations that only produce an effect under certain environmental conditions
(conditional mutations)
mutations that reverse the effect of a previous
mutation
suppressor mutations
2nd mutation in the same gene
Mutation 1 alters protein structure, 2 alters it back
Intragenic
2nd mutation in totally different gene
Mutant protein 1 is defective, mutant protein 2 does the job of protein 1
Intergenic
Types of Small gene mutations
1) Base-pair substitutions
2) Insertions/deletions
3) Expansion of trinucleotide repeats (TNRE)
One nucleotide is changed to a different nucleotide
Base-pair substitutions
Possible outcomes on the amino acid sequence with Base-pair substitutions
1) No effect
2) Change causes the wrong amino acid to be inserted
3) Change turns the codon into a stop
codon
a mutation with No effect
usually see this if the 3rd nucleotide of a codon is changed
silent mutation
Change causes the wrong amino acid
to be inserted
missense mutation
Change turns the codon into a stop
codon and Causes the polypeptide to stop growing
nonsense mutation
An extra nucleotide gets added or removed
Insertions/deletions
why are Insertions/deletions very bad
it causes a frameshift
shift in the reading frame
frameshift
Some loci contain a series of trinucleotide repeats next to a gene or inside the gene
Expansion of trinucleotide repeats (TNRE)
what Abnormal event can occur with the Expansion of trinucleotide repeats (TNRE)
Copy number increases
Abnormal DNA structure causes DNA pol to what
slip and copy section 2x
TNRE disorders usually get worse each generation
anticipation
How does DNA damage get converted into permanent mutations?
1) A change occurs in the structure of a nt (lesion/damage)
2) DNA rep occurs – DNA pol puts “wrong” nt across from the lesion
3) 2nd DNA rep occurs – Wrong nt serves as a template for complimentary wrong nt
Causes of spontaneous damage include
1) Errors of DNA polymerase
2) Tautomeric shifts
Polymerases and proofreading/repair enzymes are not what
perfect
Some major causes of spontaneous errors during replication include
a) Strand slippage (see TNRE)
b) Defective proofreading
Repeats cause abnormal loop —> DNA pol copies same thing 2x
Strand slippage (see TNRE)
a chemical reaction that occurs when the position of electrons and protons in a molecule rearrange
Tautomeric shifts
Nitrogenous bases can exist in different chemical forms called what
structural isomers
“Normal” forms
A-T, C-G bonding
“Rare” isomers
Abnormal base pairing
VERY BAD if Conversion between normal and abnormal isomers occur when
right before DNA replication
what happens if if Conversion between normal and abnormal isomers occurs at a bad time?
DNA pol will read rare form and
insert the wrong base across
A nitrogenous base shifts from the common tautomer to the rare version
tautomeric shift
Tautomeric shift steps
a) A nitrogenous base shifts from the common tautomer to the rare version
b) DNA replication begins
c) DNA replication begins again
Causes of spontaneous damage include
1) Errors of DNA poly
2) Tautomeric shifts
3) Depurination and deamination
4) Oxidative damage
5) Transposons (aka jumping genes)
Sugar-base bond is spontaneously
broken
Depurination
how is Sugar-base bond is spontaneously
broken
Base is lost (usually purines) and nucleotide
is left empty
nucleotide left empty
apurinic site
What would happen to apurinic site during
DNA replication?
can cause replication to stall or lead to mutations if bypassed, potentially resulting in single or double-stranded DNA breaks
An amino group of C or A is
spontaneously lost
Deamination
why is a An amino group of C or A is lost
- C or A w/o amino groups won’t hydrogen
bond with normal G and T - DNA pol sees a deaminated C (or A) and
puts in the wrong base
Normal process of aerobic cellular respiration creates extremely reactive
atoms called free radicals which steal electrons from DNA bases
Oxidative damage
An atom or group of
atoms that has/have an unpaired electron
Free radicals
Free radicals will steal an electron from
Proteins, lipids, DNA
what happens when this happens: Removal of electrons from DNA bases
alters their structure
Thought to be major mutagen in our cells
Cancer and aging
Mobile pieces of DNA abundantly found in all living things
Transposons (aka jumping genes)
Cut or copy themselves and then insert
randomly in the host genome
Transposons (aka jumping genes)
Transposons (aka jumping genes) encode what enzyme
transposase
move discrete segments of DNA called transposons from one location in the genome (often called the donor site) to a new site without using RNA intermediates
transposase
Insertion near genes or within genes can disrupt
host gene expression
what is control when transposase is controled
movement
Some external agents (chemical and physical) can induce DNA damage
1) Base analogs
2) Alkylating agents
3) Intercalating agents
4) UV light and low energy radiation
5) High-energy radiation (ionizing radiation)
6) Viruses
Chemicals that resemble normal nucleotides and can substitute for them during DNA replication
Base analogs
However, they exhibit abnormal base-pairing properties
Base analogs
These chemicals add an alkyl group (CH3 or CH3CH2) to
amino or ketone groups in nucleotides
Alkylating agents
exhibit abnormal base pairing
Alkylated nucleotides
Alkylating agent used as a weapon in WWI
- Soldiers came down with severe burns, blindness, and tumors
Mustard gas
what is Mustard gas an example of
Alkylating agents
Flat, multiple-ringed molecules that tightly wedge themselves between the bases of DNA distorts its 3-D structure
Intercalating agents
acridine orange and ethidium bromide are examples of
Intercalating agents
They are common used to visualize
DNA during centrifugation or gel
electrophoresis
acridine orange
and ethidium bromide
Disrupt DNA and other macromolecules
UV light and low energy radiation
λ≈260 nm and is very mutagenic
UV light
causes adjacent pyrimidine bases to fuse with one another
UV light
fused pyrimidine bases
pyrimidine dimers
Distort DNA 3-D structure
pyrimidine dimers
prevent DNA pol from replicating normally
Pyrimidine dimers
Cells containing too many of these
what will kill themselves via cell suicide (apoptosis)
dimers
EM radiation with shorter wavelengths even worse
High-energy radiation (ionizing radiation)
How High-energy radiation Mutates DNA
1) It cause electrons to be released from various
molecules in the cell producing free radicals
2) It directly breaks phosphodiester bonds in the DNA
strands (causes double- stranded breaks)
- Can produce deletions, translocations, inversions
3) Creates thymine dimers
It cause electrons to be released from various molecules in the cell producing free radicals
ionization
have the ability to randomly insert themselves into our genome
Viruses
what happens when Viruses go into a promoter or coding sequence
gene expression disrupted
produce proteins that directly inhibit DNA replication, monitoring, or repair mechanisms
viruses
can viruses be removed
no
Used to test if a new chemical has ability to mutate DNA (cause cancer)
Ames test
Ames test Set-up
- Uses bacterial strain that can’t make its own histidine (won’t grow w/o it)
- Mix bacteria w/ either chemical or H2O and add to Petri dish lacking histidine
- No bacteria should grow
*Mutations can occur to allow the bacteria to make histidine —-> regain ability to grow
Ames test results
- H2O control -»>Very few colonies (spontaneous)
- Mutagenic chemical –» lots of colonies (BAD!!)
Most types of DNA damage can be fixed by
the cell
when Must DNA damage be fixed
PRIOR TO DNA REPLICATION
what dna damage can’t be fixed
Transposons and retroviruses
Different types of DNA damage
- Altered individual bases
- Altered 3-D DNA structure
- Double-strand DNA breaks
Reverses the alteration w/o cutting out or replacing any nt
Direct DNA repair
what is Direct DNA repair used to repair
thymine dimers and alkylated bases
Both bacteria and eukaryotic cells use
light-dependent pathways
how are thymine dimers repaired
Direct repair
Eukaryotic cells – use an enzyme called photolyase to cut abnormal covalent bonds between the two thymines
- Bacteria – use an enzyme called photoreactivation enzyme (PRE) to do same
Eukaryotic cells – use an enzyme called what to cut abnormal covalent bonds between the two thymines
photolyase
bacteria cells – use an enzyme called what to cut abnormal covalent bonds between the two thymines
photoreactivation enzyme (PRE)
how are alkylated bases repaired
Direct repair: Methylguanine DNA methyltransferase enzymes directly cuts off extra CH3
from guanine
Removal of altered base/nucleotide and replacement with
good DNA
Excision repair
steps of Excision repair
- Recognition of the lesion by 1 or more proteins and the subsequent excision of that error by a nuclease enzyme
- A DNA polymerase fills in the space with proper nucleotides
- What enzyme would you predict does this in prokaryotic cells? - DNA ligase seals the final nick
2 types of excision repair systems
- Base excision repair
- Nucleotide excision repair
used for correction of minor alterations to individual bases (free radical, alklyated, base analog)
Base excision repair
steps of Base excision repair
1) DNA glycosylase enzymes recognize altered bases
2) Glycosylase then cuts out the base only (breaking the
sugar/base bond)
3) AP endonuclease enzyme recognizes the nucleotide
missing the base and makes a cut in the sugar/ phosphate backbone at that site
4) DNA pol I/ligase finish the job (and repair the damage)
fixes larger lesions that
distort the actual DNA structure and block replication
Nucleotide excision repair (NER)
steps of Nucleotide excision repair (NER)
- DNA is damaged and a lesion forms
- Proteins called Uvr (UvrA, B, C, D)
recognize the lesion and cut it out
- A-B complex recognizes the lesion
- A comes off and is replaced with C
- B-C together cut the DNA on either side of the lesion
- D is a helicase that liberates the cut piece
- DNA pol I fills in the gap/ ligase seals
what forms when dna is damaged
legion
recognize the lesion and cut it out
Proteins called Uvr (UvrA, B, C, D)
recognizes the lesion
A-B complex
comes off and is replaced with C
A complex
together cut the DNA on either side of the lesion
- Cut out extra “good” DNA on both sides
B-C
is a helicase that liberates the cut piece
D complex
human disorders exist in which the NER system is defective
xeroderma pigmentosum
Contain one of several rare mutations in some part of the NER pathway
Xeroderma pigmentosum (XP)
They have severe skin abnormalities when exposed to the sun
- UV light exposure Induces freckling, ulceration, and skin cancer
Xeroderma pigmentosum (XP)
fixes mismatches (DNA may
look okay otherwise)
Mismatch repair
“Wrong” nucleotide is always on
the new strand
Newly-made DNA strands stay
unmethylated
is always on the new, unmethylated strand
Wrong nucleotide
Mismatch repair mechanism
1) MutS protein locates mismatches
2) MutL binds to MutH
3) MutH makes a cut in the unmethylated strand
4) MutU acts as a helicase to release the unmethylated strand before an exonuclease destroys it
5) DNA pol III fills in with proper sequence, ligase seals
locates mismatches and Forms complex with MutL afterward (linker)
MutS protein
binds to MutH
MutL
bound to a nearby hemi-methylated site
MutH
DNA must loop out to allow
L-H interaction
makes a cut in the unmethylated strand
MutH
acts as a helicase to release the
unmethylated strand before an exonuclease
destroys it
MutU
fills in with proper sequence
DNA pol III
seals
ligase
Two repair pathways fix double-stranded breaks
1) Homologous recombination repair
2) Non-homologous end-joining
steps of Homologous recombination repair
a) Homologous chromosome first brought in
b) RecBCD recognizes double stranded breaks
c) RecA binds to single-stranded end and promotes invasion of the homologous chr.
d) RuvABC, DNA polymerase, and ligase help to recreate the gaps and resolve the structure
what is first brought in
Homologous chromosome ( Usually the sister chromatid)
recognizes double stranded breaks, Partially degrades 1 strand on each side, and Creates single-stranded overhangs
RecBCD
binds to single-stranded end and
promotes invasion of the homologous chr.
RecA
The good strand loops up
D-loop
help to recreate the gaps and resolve the structure
RuvABC, DNA polymerase, and ligase
The once damaged chromosome will contain a piece of the
homologous chr.
The two broken ends are simply glued back together
Non-homologous end-joining
does Non-homologous end-joining need sister chromatid
no
bind to each side of the break (to stabilize)
End-binding proteins
recruited to prevent drifting of the two pieces
Cross-bridging proteins
what happens to the ends during Non-homologous end-joining
processed, filled, and ligated
advantage of Non-homologous end-joining
Can happen any time in cell
cycle (no sister chr. required)
disadvantage of Non-homologous end-joining
Can lead to small deletions
near the break site (result of processing)
Some lesions (e.g. TT) block
normal DNA replication (via DNA pol III)
If other repair fails, what will initiate to allow DNA replication to
finish
translesion synthesis
- Stalling of normal DNA polymerase by lesion triggers
recruitment of “emergency” polymerases - Have different binding pocket more tolerant of
altered DNA structure - Emergency pols (e.g. DNA pol II, IV, V) replicate
over the lesion - Problem: They are very error prone
- DNA gets replicate, but with mistakes
- Original lesion remains (not fixed)
- Translesion synthesis enables rep to
continue
triggers recruitment of “emergency” polymerases
Stalling of normal DNA polymerase
replicate over the lesion
Emergency pols (e.g. DNA pol II, IV, V)
problem of Translesion synthesis (called SOS repair)
They are very error prone
- DNA gets replicate, but with mistakes
