Ch. 12 DNA Mutation and Repair Flashcards
What do point mutations do?
Alter one base, and possibly an amino acid.
What are the kinds of point mutations, and describe each. (5)
Transition: purine to purine
Transversion: purine ↔️ pyrimidine
Silent Mutation: codes for same a.a.
Missense Mutation: codes for a different a.a
Nonsense Mutation: adds a stop codon
What are inserts/deletions?
indels: add or remove bases and change protein length
What is a DNA lesion vs a mutation?
DNA lesion: a change in DNA sequence
Mutation: a change that is inherited
What is an open reading frame?
AUG … (XXX)ₙ … UAA/UGA/UAG
start… codons … stop
What is a frameshift?
A shift in the open reading frame caused by adding or removing one or two bases.
What happens if you add or remove three bases?
The gene will stay in frame, but there can be other negative impacts.
What can mutations in large tracts of DNA cause?
Changes in chromosome structure.
Where can these large mutations occur? (2)
Can occur in an individual chromosome or invade two chromosomes.
How can large mutations effect gametes?
May lead to errors in chromosome alignment during meiosis and crossover.
What are gene fusions?
Give an example.
Fusing of two genes that can be caused by large mutations.
e.g. promoter for one gene contralling another gene
How does water influence mutations, generally?
Being water dependent presents DNA risks due to hydrolysis reactions?
How do spontaneous hydrolysis reactions effect DNA? (3)
- Spontaneous hydrolysis reactions replace amino groups with hydroxyl groups.
- These changes in amino groups impact H-bonding with other bases
- abasic sites can occur
How do abasic sites occur?
Due to hydrolysis of the bond between deoxyribose and a base. (abasic: without a base)
How can oxidation cause mutations?
O⁻ and N⁻ containing compounds can cause oxidative damage that commonly leads to incorrect pairing.
What is the Ames test used for?
To identify possible mutagens.
What does the Ames test culture? (2 ish points)
- Mutant Salmonella (his﹣) is cultured; requires histidine to be in the culture medium.
- Random mutations will lead to his+ Salmonella
What frequently happens during the Ames test if a mutagen is present?
Reversion mutations (his﹣ to his+) occurs more frequently.
How does the Ames test actually determine if a mutagen is present? (3)
Chemical concentration is varied via a disk diffusion.
If the concentration is too high, it causes too much mutation and you get a clear zone.
If the concentration is low, it may increase mutation rate without killing the culture.
What are common cancer therapies?
DNA damaging agents.
What is cancer a result of?
Mutations.
Why can mutagens be used to treat tumor cells?
Cancer cells must divide to form tumors, so mutagens can kill them/keep them from dividing.
Why are non-dividing or slowly dividing cells not as susceptible to cancer therapies?
Non-dividing or slowly dividing cells are not as susceptible to mutagens because damage can be repaired before division.
Why do the adverse side effects of chemo occur in specific cell types?
The side effects are generally related to damage to normally fast-reproducing cells in hair follicles, blood cells, and digestive tract epithelium.
What does exposure to UV light cause? (3 ish)
UV light causes pyrimidine cross linkages. Base pairs are disrupted causing links. DNA pol stalls until repair or adds a random base.
What do higher energy waves like gamma, X-rays, and radiation cause?
How are they repaired? (2)
High energy waves cause chromosome breaks. Single break can be repaired by ligase. Double breaks are repaired in other ways.
What do tautomeric shifts in bases cause?
Tautomeric shifts in bases allow them to pair with the wrong base briefly, so 3’→5’ exonuclease misses the error.
What happens if a DNA pol slips?
A pol can slip on a repetitive base sequence, causing an indel.
Are all pol’s the same fidelity, what does this cause?
No. Not all pol’s have a fidelity as high as the primary replication pol which can cause errors.
What are errors in homologous recombination caused by?
They are due to repeated regions on homologous or non-homologous chromosomes.
Why must DNA repair occur?
DNA integrity must be maintained for a species to proliferate generation to generation.
Why do molecular biologists suggest DNA repair mechanisms have an origin early in evolutionary history?
There are multiple mechanisms of repair, but some are found in all cells with little divergence.
What is the mismatch repair system?
MMR: one mechanism of DNA repair that is well studied in prokaryotes and preferentially repairs daughter strand DNA.
How many proteins facilitate MMR?
multiple proteins
How does MMR repair DNA? (6)
- MutS/MutL recognizes a DNA lesion on newly replicated DNA and binds
- MutS/MutL forms a loop to scan the DNA adjacent to the DNA lesion until it finds a GATC site (Dam methylase site)
- MutH is recruited to the complex
- MutH cuts the unmethylated GATC sequence to create a nick
- a helicase binds and displaces DNA containing the error, which gets degraded
- DNA pol binds and replaces the removed bases
Is MMR energy intensive and why does it imply?
It is energy-intensive and illustrates the importance of maintaining genome integrity. Uses ATP because motor proteins are moving along DNA.
Is MMR a direct or indirect repair mechanism?
indirect
What is photolyase?
A repair mechanism that occurs in most cells except those in placental mammals.
How does photolyase work? (3)
- photolyase absorbs a photon and passes a high energy electron to excite FADH-
- FADH- passes and electron to a pyrimidine dimer to reverse the crosslinks
- the electron passes back to photolyase
Is photolyase a direct or indirect repair mechainsm?
It is direct; it doesn’t cut phosphodiester bonds.
What are methyltransferases?
Enzymes that repair some improperly methylated bases by removing the methyl group. It is a DNA repair mechanism. (Humans have these!)
How do methyltransferases work? (2)
- methyl group transfers to the enzyme, which is then degraded
- sacrifices an enzyme (methyltransferase) to fix one base
What does the sacrifice of a methyltransferase indicate?
The sacrifice of an enzyme to fix one base illustrates the importance of maintaining the genomic integrity.
Are methyltransferases a direct or indirect repair mechanism?
direct; it doesn’t break phosphodiester bonds
What is base excision repair?
A repair mechanism that recognizes and repairs damaged nucleotides and single strand breaks.
How does base excision repair work? (1,2,3a, 3b)
- a DNA glycosylase recognizes damage and removes the base from the nucleotide
- AP endonuclease makes a nick next to the site
3a. in bacteria, DNA pol binds and displaces the error AND adjacent bases; ligase seals nick
3b. in eukaryotes, a short repair is most common, but long repairs can also occur
Are DNA glycosylases generic or specific?
What is the example from class?
They are specific. Different glycosylases are used for different base errors.
uracil DNA glycosylase (UDG): removes uracil from DNA nucleotides creating an abasic site
Is base excision repair a direct or indirect repair mechanism?
indirect
What is nucleotide excision repair?
NER repairs large lesions
What proteins is NER mediated by in bacteria?
Uvr proteins
How does NER occur in bacteria? (4 )
- UvrAB recognizes a lesion and attracts UvrC which has exinuclease activity
- Exinuclease cuts the eighth phosphodiester bond to the 5’ side of teh lesion and the fifth phosphodiester bond to the 3’ of the lesion generating a 12-13 nt fragment
- UvrD, a helicase, binds and removes the fragment
- pol I fills the gap and ligase seals the nick
What proteins is NER mediated by in eukaryotes?
XP proteins
How does NER occur in eukaryotes? (3)
- XPC recognizes damage and recruits XPBD which are helicases that form a bubble
- XPF and XPG binds and create 5’ and 3’ nicks, creating a 24-32 bp fragment
- DNA pol fills the gap and ligase seals the nick
What happens to NER in eukaryotes if it is stalled at a lesion?
transcription-coupled repair: when RNA pol can also repair DNA if stalled at a lesion
Bacteria have a similar mechanism.
Why must chromosome breaks be repaired?
Chromosome breaks can disrupt a replication fork and MUST be repaired for a cell to divide.
What are the two recombination mechanisms used to repair DNA breaks?
Homologous recombination (HR)
Non-homologous end joining (NHEJ)
What does homologous recombination require?
HR requires a homologous chromosome to repair chromosome breaks.
What is NHEJ and when is it used?
NHEJ can also be used to repair chromosome breaks. It is used when homologous recombination is not an option.
What is a slim, but possible outcome of NHEJ?
There is a potential for joining fragments of different chromosomes, but it is small.
Why are errors in ssDNA of note?
If an error in ssDNA at a replication fork is present, a complementary base is not available to guide repair.
What is translesion synthesis?
Something some pol’s exhibit; the ability to read across and error to allow continued DNA replication. This can fix the issue of a ssDNA error at a replication fork.
What is the downside of translesion synthesis? (2)
- pol’s lack 3’→5’ exonuclease activity
- often results in mutation
If there are downsides of translesion synthesis why do it?
It is a desperation strategy to allow replication to continue at the cost of genome integrity.
No replication=cell death
