12 mutation and repair slides (exam 2) Flashcards
A mutation is:
changes in DNA sequence
When are mutations heritable?
- Single-celled organisms
2. Germ-line cells of multicellular organisms
What are the consequences of mutations?
- Neutral = most common
- Harmful
- Beneficial = least common
- Mutations may provide variability for natural selection
T/F Cells have an unlimited amount of time to remove mutations:
False, they have a very limited amount of time before the mutation gets replicated and embedded into the genome.
Fewer than ______ damaged nucleotides become mutations
1/1,000,000
Change in dna through generations can result in either a
- loss of function
- gain of function
What are 3 examples of a loss of function mutation?
- destroy enzyme active site
- Truncated proteins
- Disrupt gene expression
What are 3 examples of a gain of function mutation?
- Increase enzyme affinity
- Remove regulatory regions of proteins (proteins are less regulated)
- Increase gene expression
What is a point mutation?
Change in a single base pair
What are the 2 types of point mutations?
- Transitions
- Purine –> purine
- Pyrimidine –> pyrimidine - Transversions
- Purine –> pyrimidine
- Pyrimidine –> purine
What are the possible consequences of point mutations? (3)
silent mutation
missense mutation
nonsense mutation
what is a silent mutation?
amino acid isn’t changed therefore mutation does not present itself
What is a missense mutation?
single base pair mutation changes the amino acid into something different
What is a nonsense mutation?
single base pair mutation change amino acid into early stop codon and ends transcription
2 steps of point mutations:
- mismatch is incorporated
2. replication fixes the mismatch in the next generation
what are oncogenes?
genes that drive cell division forward
- gain of function leads to cancer
- mutation usually dominant
what are tumor suppressor genes?
- genes that stop cell division
- Loss of function leads to cancer
- Usually recessive mutation
- AA = normal
- Aa = predisposition to cancer
- aa = cancer
what are indels?
insertions/deletions
What happens with indels?
indels of 1-2 nucleotides lead to frame shifts.
- Indels of 3 nucleotides (codon) preserve reading frame but remove or add amino acid
What causes triplet expansion diseases?
Template slippage
What happens during template slippage?
- In the first step, DNA polymerase encounters the direct repeat during the replication process.
- The polymerase complex suspends replication and is temporarily released from the template strand.
- The newly synthesized strand then detaches from the template strand and pairs with another direct repeat upstream.
- DNA polymerase reassembles its position on the template strand and resumes normal replication, but during the course of reassembling, the polymerase complex backtracks and repeats the insertion of deoxyribonucleotides that were previously added. This results in some repeats found in the template strand being replicated twice into the daughter strand. This expands the replication region with newly inserted nucleotides. The template and the daughter strand can no longer pair correctly.[4]
- Nucleotide excision repair proteins are mobilized to this area where one likely outcome is the expansion of nucleotides in the template strand while the other is the absence of nucleotides. Although trinucleotide contraction is possible, trinucleotide expansion occurs more frequently.[2]
What is the disease that has CAG trinucleotide expansion?
Huntington’s Disease
What are many chromosomal rearrangements caused by?
- homologous and non-homologous recombination (crossing over in prophase I of meiosis)
What are the chromosomal rearrangements found in a single chromosome?
- deletion
- duplication
- inversion
What are the chromosomal rearrangements found in multiple chromosomes?
- Insertion
* Translocation
What happens during deletion?
May cause:
•Complete loss of genes
•Bring genes into proximity
What happens during duplication?
May cause
•Increased gene dosage
What happens during inversion?
May cause
•Disruption of genes
•Bring genes in proximity
What happens during insertion?
DNA exchange between the telomeres of chromosomes
What happens during translocation?
- Exchanging the ends of chromosomes
- Remember human chromosome 2 and chimpanzees?
- Also cause of rare form of Down syndrome
What type of translocation causes a rare form of down syndrome?
Robertsonian translocation
What is a fusion gene?
•Translocations and insertions can lead to fusion genes•Hybrid of 2 different genes•Genes that are fused and lead to upregulation or downregulation of a gene
what is damage by water?
hydrolysis
What does hydrolysis lead to? (2)
- deamination
- depurination
what is hydrolysis?
cleavage of a molecular by water
What does depurination lead to?
abasic sites
DNA has a negative charge from the ______ _______.
phosphate backbone
ROS stands for?
reactive oxygen species (
ROS:
- nitrous acid induced deamination
- oxidative damage
What do alkylating agents cause?
Damages by alkylation
What is alkylation?
Adding chains of carbons or other organic molecules
What is the chemical name for mustard gas?
mechlorethamine
What does chemotherapy technically do to DNA?
Intentionally damage it
What cells does chemotherapy ultimately target?
- actively diving cells.
This explains why other normal rapidly dividing cells are affected. - (hair follicles, blood cells, digestive tract)
Why are people that survive primary cancer at an increased risk of a secondary tumor?
Chemotherapy can cause unintended mutations
What does cisplatin do?
- alkylating agent
- between purine N-7’s
- stalls replication fork
What does Bleomycin do?
- direct OH radical damage of DNA
What does doxorubicin do?
intercalates – inserts between bases leading to double-stranded breaks
what 2 type of ionizing radiation lead to dna damage?
- UV damage
2. Gamma rays and x rays
How does UV damage change the DNA?
- causes pyrimidine dimers (adjacent thymines)
- cyclobutane
- 6-4 photoproduct
- -ultimately kinks DNA making it more susceptible to damage
How do gamma rays lead to dna damage?
By causing single and double stranded breaks.
what does a single stranded break do?
causes a nick in one strand and fragments one chromosome
How does a DSB influence the chromosome?
causes nicks in two strands, fragments both daughter chromosomes
DNA damage is _____ and _____
diverse ; complex
what are the two main types of dna repair?
- direct repair
2. excision repair
What is direct repair?
repair/reversal via an enzyme that doesn’t break phosphodiester backbone.
what is an example of direct repair?
1) Photolyase
2) Alklytransferases
What is excision repair?
removal of damaged DNA and replacement by complementary template.
What is an example of excision repair?
1) MMR
2) BER
3) NER
4) DSBR
What is the direct repair mechanism via photolyase?
- Bacteria produce photolyase
- Photolyase repairs UV damage (pyrimidine dimers)
- Utilizes the electron carrier FADH
- Photolyase is present in almost all organisms except mammals
Is photolyase present in mammals?
No, but it is present in almost all organisms besides them
What electron carrier does photolyase utilize?
FADH
What does photolyase repair?
UV damage (pyrimidine dimers)
What produces photolyase?
Bacteria
What is the direct repair mechanism via alkyltransferases
- Remove alkyl groups that tend to cause nonstandard base pairing
- Basically use a base to pull alkyl groups from another base
- Example: methyltransferase
- Used up in the reaction= “suicide enzyme”*
- Must be degraded
What are enzymes that are used up in a reaction?
suicide enzymes
What happens in excision repair?
- removes bases
2. replaces bases with the aid of template strand
What are some examples of excision repair?
- MMR
- BER
- NER
- DSBR
What happens during mismatch repair (MMR)?
- For repairing mismatched nucleotides and indels up to 3 nt
- Mismatched bases causes a detectable distortion
- Repair usually based on parent strand
What is the mechanism for MMR in E. coli?
- In E. coli, new strand is recognized by hemimethylation
- Parent strand is methylated, new strand is not
- A distortion of DNA is detected on new strand by MutS-MutL
- MutH cleaves at unmethylated site
- 5’ to 3’ or 3’ 5’ exonuclease and helicase removes DNA
- DNA replaced with Pol III and ligase
What is MMR recognized by in E. coli?
Hemimethylation
What is MMR mechanism in eukaryotes?
New strand may be detected by nicks in the new strand between primers
• MSH2 and MSH6 in yeast = MutS-MutL in E coli.
• Doesn’t appear to require helicase
• Damage to MMR genes lead to HNPC/Lynch syndrome • Colorectal cancer
What system does E. coli use for MMR?
MutS-MutL
What system does yeast use for MMR?
MSH2 and MSH6
Does eukaryote MMR require helicase?
It doesn’t appear to, No.
What does damage to MMR genes lead to in eukaryotes?
HNPC/Lynch syndrome
- colorectal cancer
What is base excision repair?
- Single damaged nucleotide or
2. Single-stranded breaks (SSBs) that need “cleaning” (can’t ligate to nearby strand)
What is the most prominent form of DNA repair?
Base excision repair (BER)
How does BER occur in bacteria?
- DNA glycosylase cleaves base at 1’C of the sugar
• Specific DNA glycosylases recognize different types of damage
• Flips out damaged base—causes AP site (apurinic or apyrimidinic) - DNA backbone cleaved by AP endonuclease
- Base replaced by DNA PolI and ligase
What is an AP site?
Site with no base
What cleaves the backbone at AP sites in prokaryotes?
AP endonuclease
In prokaryotic BER, what cleaves the base at the 1’ C of the sugar?
glycosylase
T/F specific DNA glycosylases recognize different types of damage
True
What replaces the base in BER in bacteria?
DNA poly I and ligase
What are the steps of BER in eukaryotes?
Same first 2 steps as in bacteria
(1. DNA glycosylase cleave base at 1’C of the sugar
• Specific DNA glycosylases recognize different types of damage
• Flips out damaged base—causes AP site (apurinic or apyrimidinic)
2. DNA backbone cleaved by AP endonuclease
But THEN.
Long patch or short patch
• Long patch = for long stretches of DNA; uses flap endonuclease
• Short patch = for repair of single nt base; uses Pol β
what enzyme is used in long patch BER in eukaryotes?
Flap endonuclease
What enzyme is used in short patch BER in eukaryotes?
uses Pol β, for repair of single nucleotide bases
What does NER stand for?
Nucleotide excision repair
How do mammals repair pyrimidine dimers?
(NER) nucleotide excision repair, because they do not have photolyase
How does NER work?
- Recognizes bulky lesions (alkylation and UV damage)
* Removes DNA on both sides of damage site via excinuclease
What removes DNA on both sides of damage in NER?
Excinuclease
What are 2 examples of bulky lesions?
- alkylation
2. uv damage
How does NER occur in bacteria?
- Recognized and repaired by UVR proteins
- Recognize bulky lesion
- Single stranded bubble created
- 5th phosphodisester bond on 3’ side of lesion cleaved • 8th phosphodisester bond on 5’ side of lesion cleaved • Filled by Pol I and ligase
NER in eukaryotes:
Similar to bacteria, but with differently named proteins
Unrepaired pyrimidine dimers may lead to ______
Melanoma
Mutations in the NER system lead to
xeroderma pigmentosum
What are SSBs
single stranded breaks
What are DSBs
double stranded breaks
What is the deadliest form of skin cancer?
Melanoma
DSBR stands for what?
double stranded break repair
DSBs may result in…
- loss of the whole chromosome sections
- usually caused by ionizing radiation
How are DSBs fixed?
HR (homologous repair)
OR
NHEJ( non homologous end joining)
What happens during homologous repair?
Damaged DNA uses an intact template strand to replace DNA
What happens during Non-homologous end joining?
If an intact template is unavailable, enzymes will bind, trim and ligate damaged dsDNA
