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What is the difference between a mutation and a DNA damage/mismatch?
DNA damage (including chemically errors such as broken or missing base)/ mismatches are transient mistakes in the DNA
Mutations are stable, heritable (on a cell level), changes in the DNA. They are changes in the sequence of DNA on a base pair or chromosomal level (correctly paired, but a different sequence than the original)
DNA damage becomes a mutation when the alteration is converted through replication to correctly paired, but incorrect base pair sequence
What are the three potential sources (mutagenic processes) of mutations in DNA?
DNA replication- low but significant error rate
Spontaneous DNA damage (chemical changes to DNA ex imino vs amino form of nucleic bases)
DNA damage caused by attack from outside mutagenic agents
What is the difference between if a mutation occurs in somatic cells vs germ line cells?
Somatic cells are every other cells in your body except egg, sperm and precursor cells. If mutations occur in these cells somatic cells that bear that mutation only affect the parent
If a mutation occurs in germ-line cells entire generation could be effected as the mutation become heritable. Effects somatic and/or germ-line cells of 2nd generation!
What is a point mutation? What type of error is it and how does it turn into a mutation?
Change in a single base
a two step process that is a replication error. In the first generation there is a mistake match/ failure to proofread and lead to a base pair mismatch. In the second generation the replication error becomes a mutation as the point mutation change is in one of the replicated DNA molecules (but now correctly paired with the proper base) for future division whereas the other sequence has the normal sequence
What are the two types of errors that result in mutations in DNA and what proteins are responsible for “fixing” these errors?
Replication errors - proofreading (DNA polymerase E and delta) and mismatch repair (MMR)
DNA damage - other DNA repair mechanisms
What is the difference between transition and transversion base changes in point mutations?
Transition: change of bases in the same family. Purine flipped with another purine or pyrimidine (G-A) exchange with an other pyrimidine (C-T)
Transversion: interchanged of family so purine to pyrimidine an exchange between one ring and two ring structure
What are the effects of a point mutation?
Silent mutation- in gene sequence that does not effect the amino acid sequence coding for the protein (such as a mutation to the promotor region)
Missense mutation - DNA change that results in different amino acids being encoded at a particular position in resulting protein
Nonsense mutation - a change in DNA that causes a protein to terminate or end its translation earlier than expected
How is a frameshift mutation different than other point mutations?
Frameshift mutations are the results of indels (either insertions OR deletions of bases) and can be multiple
ex: a deletion of 1 amino acid will result in a shorter probably nonfunction protein in which codons are shifted by 1
can be deletion or addition of 1 or 2 amino acids, but indel of three would not cause a frameshift mutation as the original code sequence integrity would be maintained with the addition or deletion of a entire amino acid.
What is the purpose of mismatch repair and what is the mechanism?
MMR corrects normal nucleotides in incorrect base pairs
if there is an error in the newly made strand mismatch proofreading proteins mutSa and mutLa bind to the mismatch. Mutsa scans (and is always scanning) the DNA for the mismatch mutLa loops the DNA until it finds a nick (DNA that has not yet been attached by ligase such as Ozakazi fragment or methylation pattern) then cuts out a new strand for DNA polymerase to reduce
What is a microsatellite and why are they dangerous?
Regions of the genome with short repeating sections of nucleotides that can lead to polymerase slippage and indels
What is endogenous DNA damage?
Spontaneous something that is already in your body is causing the damage
make a far greater contribution to genome mutations but DNA shields potentially reactive groups of bases to provide some protection
Damage is TRASCIENT mutations are PERMANENT
What type of damage is depurination?
A purine base is removed from the DNA
Apurinic/ apyrimidinic (AP site) is a location in the DNA where there is no base. The base is lost but the phosphate and sugar group is maintained therefore there is no coding information
What is deamination and how does it affect DNA? Which base can not be deaminated?
The removal of an amine group (R-NH2) which can create a mutation if not fixed can be caused by water
EX: Cytosine pairs with guanine but after deamination it becomes a uracil which pairs with adenine
Thymine does not have an amine group and therefore can not be deaminated.
Why would a deamination of 5-methylcytosine to thymine result in the most mutations?
Thymine is normal in DNA and therefore would not raise any red flags or look weird. More likely to be accepted.
Why does the oxidation of bases in DNA cause damage?
Oxidation loss of electrons in bases of DNA which they do not like come from reactive oxygen species from oxygen in your mitochondria
deoxyguanosine (dG) → 8-oxo-deoxyguanosine (8-oxo-dG) which binds with Adenine (conversion after replication)
What type of damage can reactive oxygen species cause?
oxidation of bases in DNA
AP sites (no base)
DNA strand breaks (single or double) → species with higher respiration rate have higher rates of ROS
Why are anti-oxidants important in your diet?
They scavenge and remove reactive oxygen species in your body that can cause DNA strand breaks, oxidation, and AP sites
What is the example of exogenous DNA damage?
Environmental DNA damage such as carcinogens (physical and chemical)
ex: X-rays strip electrons from water molecules and create ROS resulting in single and double DNA strand breaks
How does UV-induced DNA damage effect base pairs?
UV radiation is blocked by the ozone layer
Two pyrimidine bases will get covalently bonded to each other instead of connecting by hydrogen bonding next to each other not across (typically either cytosine or thymine) and form a cyclobutane pyrimidine dimer (CPD) or 6-4 photoproduct
Highly mutagenic because DNA polymerase does not know what to do when it gets to this location as neither product can be placed in its active site and bases can not be added across from it
What is a characteristic of many skin cancers?
CC to TT mutations caused by UV-radiation that is rare in other cancers and not found in skin cancers on the bottom of your feet that does not get exposed to much sun.
What is known as the incidence of squamous cell carcinomas?
Increase 10-fold every 10 degrees latitude closer to the equator
How does DNA alkylation affect DNA and what are some exogenous causes?
Add affecting alkyl groups on DNA base pairs (such as 1-methyladenine)
Mustard gas
Alkylating agents are often used experimentally to induce tumors in laboratory animals as they are among the most potent mutagens
How can chemicals become carcinogens after metabolic processing?
the liver attempts to break down pre-carcinogenic material and accidently makes it carcinogenic instead of detoxifying it forming DNA adducts
How does benzopyrene become carcinogenic?
Benzopyrene is a procarcinogen in tobacco and in attempting to detoxify it (Cytochrome P450) benzopyrenediolepoxide (BPDE) is created and is a carcinogen. It binds to DNA with a covalent bond and due to its bulky structure during DNA replication it will stall it and the cell will either die if it cannot complete replication or it will cause a mutation to avoid death.
How does Aflatoxin B and heterocyclic amines form DNA adducts?
Found in infected grain or peanuts fungally infected by Aspergillus
aflatoxin B1 becomes toxic after cytochrome P450 attempts to fix it can either be
reversed by epoxide hydrase and become detoxified
turned into a DNA adduct due to an alkylating agent addition and attaches to DNA
glutathione S-transferase can detoxify aflatoxin B a different way
Where does heterocyclic amines come from?
Combustion products (such as burnt red meat) creates PhIp which causes DNA adducts
How does mutational spectra give clues to identify the cause of the carcinogen?
Different cancers from different carcinogens have particular types of patterns of cancer mutations
EX: BP (benzopyrene) most frequently causes G:C pairs to convert to T:A pairs (G→T) transversions and in the spectra in lung cancer with smokers and coal factory workers this mutation frequency is higher than lung cancer of non-smokers
What is the difference between driver and passenger mutations in cancer?
Driver mutations are targeted in therapies and contribute actively to cancer they help the cell grow, survive and move
Passenger mutations occur because of the high mutation rate within cancer cells but no actually effecting the cancer positively
How does melanin protect cells from mutagen attack?
Melanocytes (skin cells) make and produce melanin in melanosomes. Melanin is then given to keratinocytes that convert melanin into supranuclear caps (sun umbrellas) and they are placed above on above the cell to protect the nucleus
What are the three types of DNA repair mechanisms to minimize genetic damage?
Direct Reversal
Excision repair (NER, BER) → cut out and replace
Break repair (NHEJ, HR(HDR)) → deal with DNA break
What type of repair does MGMT do?
Direct reversal of base alkylation by O6-methylguanine-methyltransferase. The alkylated G flipped out of double helix and it is directly removed
What are the two types of excision repair?
Base excision repair- repairs lesions largely from endogenous sources such as ROS and AP sites that form non-distorting lesions
Nucleotide excision repair- removes large lesions caused by exogenous agents that distort the DNA double helix
How does short base excision repair (BER) function?
When a chemically altered base causes a little helix distortion DNA glycosylase is recruited and cuts out the damaged base leaving an AP site. AP endonuclease and lyase take out the rest of the backbone. B+ DNA ligase puts a missing base back in the whole (nucleotide inserted, closure by ligase) without the need of a primer
How does long patch base excision repair (BER) function?
When a chemically altered base causes a little helix distortion DNA glycosylase is recruited and cuts out the damaged base leaving an AP site. AP endonuclease and lyase take out the rest of the backbone. Additional base pairs peel into a flap by a flap endonuclease and leave a hole. New base pair addition is initiated by pol-B (low processivity, inaccurate, and accepts very small gap) and strand displacement by pol-S or e which finishes longer sequences. DNA ligase then seals the strand.
How does nucleotide excision repair (NER) function?
Has to be a chemically damaged base that is big enough to distort the double helix (helix distorting adduct) then DNA fragment containing adduct will be cleaved 24 nucleotides on 5’ side and 5 on 3’ side. Then normal replication machinery will be recruited and pol-S or e with PCNA and RPA will fill in and ligase will close it.
What are the two pathways of NER?
Transcription coupled: follows RNA polymerase around and makes sure lesions of DNA are fixed before transcription occurs
Gene Maintenance: Scans genome for areas not open from transcription and fixes
Most proteins start with XP (named after a specific cancer)
How does homology directed repair (HDR) repair double stranded DNA breaks?
When a double stranded DNA break occurs you want to make sure the proper ends are aligned to be added back together. Therefore resection by exonuclease will remove small sections of each stand then covered by RPA and will look for information from homologous chromosomes or sister chromatid (with Rad51) to see if base pairing with unwound DNA can be used to orient broken DNA ends. The strands are then extended based on the homologous chromosome template before they are disengaged and paired. The gaps are filled and ligate restore wild type helix.
How does non-homologous end joining (NHEJ) repair double-stranded breaks? What is important to remember about it?
Conducted when HDR cannot be done because an available homologous chromosome or sister chromatid is not available. It fixes DSB in an error-prone manner because it basically just guesses
DSB then resections of single strands by exonuclease, DNA strands brought together attempting to find some complementarity and then strands are filled in and joined by ligation.
How is translesion synthesis (Pol Switch) different than repair mechanisms?
It is a special mechanism to deal with DNA damage not repaired before replication.
When there is a DNA break during replication Pol-S wants to be so perfect that it simply gives up and breaks off. The PCNA is then ubiquinoned and Rev 1 and Pol-Z are recruited and it either places something across from the damage and then extends the DNA (only polymerase that can extend the lesion) or it guesses taking a temporary piece of damage and makes it a permanent mutation different polymerase finishes strand
Why is Pol-Z special?
It along with other special polymerases have special mechanisms to deal with DNA damage not repaired before replication, but risky. They have an active site that is more open and can accommodate a nucleotide and the lesion and they can recognize specific types of damage and are more or less error-prone (but none have proof reading ability)
Pol-ada can place AA across from T T dimers
What is the difference between chromosomal instability (CIN), and microsatellite instability (MIN)?
CIN - chromosomal instability (changes in chromosome number and structure; wrong number, translocations, mismatched wrong colors)
MIN - microsatellite instability (point mutations and multiple small mutations but the chromosomes appear normal)
BOTH CIN and MIN are sufficient on their own to contribute to tumorigenesis and are not always mutually exclusive
How do defects in the exonuclease activity of pol delta and e lead to cancer?
With one amino acid substitution one gene of the set may loss the ability to proofread, if both genes are mutated in mice 75% die
In humans germline mutations in exonuclease domains of Pol-delta and Pol-E predispose families to colon cancer
How does MGMT help to protect against cancer? What was the study that proved this?
MGMT reverse alkylation damage
When mice were exposed to MNU (potent alkylating agent) mice with wild-type MGMT got lymphoma while those with increase expression (beyond normal amount) were immune to lymphomas
MGMT is silenced in 40% of gliomas and 25% of lung/lymphoma/head and neck cancer
Explain why Xeroderma pigmentosum (XP) patients have 1000 fold increase risk of skin cancer.
Loss of any protein in NER or Pol-ada results in the inability to fix UV-damage that causes large damaged regions
What do MMR defects lead to?
Mismatch repair proteins that also help decrease slippage lead to microsatellite instability that can cause cancer
cannot fix replication patterns there they have the highest mutation in genome as they can not even fix mistakes on passenger mutations (accelerate mutations)
hereditary non-polyposis colon cancer (HPNCC) arises from defects in MMR
What repair do BRCA1 and BRCA2 proteins participate in?
Double stranded DNA breaks (purpose unknown)
BRCA 1 localizes to the site of double stranded breaks
BRCA 2 partial loss of function results in chromosomal aberrations (funky chromosomes such as three chromosomes together) and more double stranded breaks
Genes are “caretakers” involved in maintenance of genome integrity
How is the tissue organized to protect stem cell genome?
Stem cell compartment is in tissues that need to be regenerated and need stem cells to regenerate in specialized cells to replenish (such as gut cells with high turn over rate) and they are shielded from toxic agents
A stem cell only completes mitosis once and typically one cell will remain a stem cell one will become a transit-amplifying cell which is supposed to divide like crazy into highly differentiated (exposed to toxic agents) cells before cell death occurs due to high turn over.
What are the two levels of protection from stem cells?
Anatomically protected from toxic agents (physical shields)
Infrequent cell division
How is division of stem cells “based on need?”
Typically a stem cell will divide and 1st daughter cell is a stem cell that will eventually replace stem cell while other is transit-amplifying cell
However depending on need stem cell can be triggered to divide differently
both can be stem cells if they need to repopulation or both transit amplifying cells
