MCC Wk 2 DNA Repair & Variance Selection Flashcards
4 major DNA repair pathways
- BER = base excision repair
- NER = nucleotide excision repair
- Double Strand Break Repair (2 subtypes):
a. HR = homologous repair
b. NHEJ = non-homologous end-joining - MMR = Mismatch repair:
a. polymerase proofreading (only during replication)
b. MMR = mismatch repair (anytime)
What is the mutation frequency?
~60 mutations per cell division (mutation frequency ~10^-8/base pair)
What 3 DNA repair pathways are error prone?
- NHEJ (double-strand breaks) = jam the broken ends back together and hope for the best
- Translesion synthesis (damage on one strand of the DNA during copying) = bypass bad stretches of template during DNA synthesis by low-fidelity copying)
- BER =series of pathway w/ overlapping substrates, some error-prone, others error-free
What 4 DNA repair pathways are mostly error prone?
- BER w/ high fidelity polymerases
- NER
- MMR - mostly done during replication
- Homologous recombination for DSBs or one damaged strand during copying = find another (from paternal or maternal) copy of the damaged sequence and copy it)
What types of DNA damage are mutagenic?
Potentially all - no form of repair is fully w/o risk
-cancer is associated w/ increased error-prone repair and decreased error-free repair
What are 4 types of DNA damage/abnormalities?
- nicks
- DSBs
- bulky lesions
- mismatched bases
What are 6 common features of DNA repair pathways?
- sensors to detect damage
- nucleases (glycosylases for BER) - remove damaged base w/ a glycosylase, then break phosphodiester backbone to allow resynthesis
- polymerases - fill in gaps in DNA as the primer for synthesis
- ligases - seal nicks in phosphodiester backbone
- helicases - unwind DNA from the nick
- recombinases - find an intact version of the damaged info and initiate strand invasion (only applies to homologous recombination)
When do most mismatch errors occur?
During DNA replication b/c of base recognition errors
When does most MMR (mismatch repair) occur?
During replication
What are restriction enzymes?
an enzyme made mostly by certain bacteria, can cleave DNA at or near a specific sequence of bases
What DNA repair fixes UV radiation induced mutations?
nucleotide excision repair
What are deoxynucleotide triphosphates (dNTPs)?
The building blocks of DNA, which include deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate, (dTTP), deoxycytosine triphosphate (dCTP), and deoxyguanosine triphosphate (dGTP)
What lab technique requires the addition of all 4 dNTPs?
PCR
What does the DNA polymerase complex contain that acts as a “delete key” for MMR?
A 3’->5’ proofreading exonuclease domain that can remove misincorporated nucleotides and correct DNA polymerase errors that occurs immediately after DNA synthesis
-deletes error
-corrects ~99% -> ~600 errors remain
What is the difference b/w BER and NER?
BER = base excision repair
NER = nucleotide excision repair
NER is for larger, helix-distorting damaged areas by UV rays, radiation, mutagens (exogenous)
BER for smaller, do not distort helix (1-6 bases) repair - endogenous (alkylation, deamination, oxidation)
What is the difference b/w DNA polymerase η (eta) and Pol z (zeta)?
DNA polymerase η (eta) always inserts “AA” for a distortion of “TT” and is a product of XP-V gene (xeroderma pigmentosum variant) which makes people highly sensitive to UV damage
Pol z (zeta) - add random bases. Quick fix but error prone
What does MSH2/6 do?
A protein that joins with another protein called MSH2, forming a complex that IDs location on DNA where errors have been made during DNA replication
What is a protein that operates in MMR?
MSH2/6
-helps recognize mismatches of various size
-loss of this -> inability to repair mismatches of different sizes -> MSI phenotype (micro satellite instability)
What induces double-stranded breaks?
Ionizing radiation
What are 2 repair pathways for DSBs?
NHEJ = non-homologous end joining
HR = homologous recombination
What is the Holliday junction?
Formed during Homology-dependent repair (HDR or HR)
-> recombination of the 2 chromosomes ~50% of the time
-Crossing over during HR leads to LOH distal to the site of repair in the next mitosis
What is translesion synthesis?
Pathway of DNA repair that involves DNA polymerases that facilitate DNA replication (thus cell division) by efficiently bypassing various DNA lesions. The damage is not removed.
-mutations are introduced and errors in the template remain. Damage needs repair later.
What BER enzyme removes nucleotides at the 3’ end?
lyase
What BER enzyme removes nucleotides at the 5’ end?
AP-endonuclease
Compare ssDNA and dsDNA repair pathways
When in the cell cycle do BER, MMR, and NER occur?
BER - anytime
MMR - mostly in S
NER - G1
What are base-specific glycosylases?
In BER. remove the altered base and create an AP (apurinic/apyrimidinic) site
ex. cytosine deamination is repaired w/ uracil glycosylase
What repair pathway allows for restart of stalled replication forks?
Translesion synthesis - if a replication fork reaches the damage site (or any unreadable damage) then error-prone translesion sythesis takes over, but also makes mutations
-mutations are introduced AND the errors in the template remain after the fork passes
What is PARP?
PARP (poly ADP ribose polymerase) is an enzyme that helps repair DNA when it’s damaged by recruiting BER machinery to nick and complete DNA repair
What are PARP inhibitors?
Pharmacological inhibitors of the enzyme PARP indicating from the tx of heritable cancers since some are more dependent on PARP than normal cells
What happens if a replication fork reaches the nick caused by BER (the initial step in BER repair)?
The nick becomes a DSB, which requires either PARP activity or HR
*loss of PARP and HR is lethal
What is a consideration if cells lack HR due to mutations in BRCA1/2?
They are sensitive to PARP inhibitors because either PARP or HR are needed for survival if a BER nick becomes a DSB. Loss of both is lethal
What are 8 causes of DNA damage?
- spontaneous hydrolysis of bases (life in water) -> abasic sites repaired by BER
- hydrolysis (deamination common)
- alkylation (methylation) alters bases and their pairing partners
- UV radiation causes adjacent pyrimidines in DNA to fuse -> dimers
- intercalating agents (esp ones that are also alkylators) -> multiple types of mutations, incl frameshifts (insertion/deletion mutations)
- Psoralens in plant-based foods are bifunctional alylator/intercalators
- Platinum derivatives -> interstrand & intrastrand crosslinks
- oxidation damage (X-rays cause same chem as oxidation b/c X-rays cause water to form free radicals)
What are 5 potential sources of mutagens in food?
- psoralens (plant defense compounds)
- antibiotics found in molds
- byproducts of cooking (roasting, charring)
- byproducts of preservation (nitrosoasmines in pickled vegetables)
- pesticide residue
What are 3 mutagens in the air we breath?
- smoking - nitrosoamine, free radicals, intercalators/alkylators
- inhalation of radon
- pollution
What are 5 mutagens from background exposure?
- oxygen (resp frequency, free radical formation)
- UV radiation (sun)
- gamma radiation/radioactive materials (incl radon)
- cosmic rays (increased at high elevation, air travel)
- cosmetic compounds (dyes)
3 mutagens encountered during medical procedures
- X-rays, CT
- radioactive tracers (PET scans)
- radiation therapy (X-rays, brachytherapy w/ alpha-emitters)
3 mutagens from smoke (cigarettes, campfires, house fire, etc)
- nitrosamines -> alkylation
- benzo[a]pyrene -> intercalation, alkylation
- free radicals -> oxidative damage
What are the 3 main sources of ionizing radiation exposure?
- radon
- background radiation (cosmic rays, radioactive materials in envir)
- medical diagnostic methods
How many alleles do histones have?
1 allele in human population
- no changes tolerated at level of viability (not b/c they don’t mutate but b/c natural selection doesn’t allow variation in the genes)
What does selective pressure dictate?
Whether variants in a gene are tolerated. Selection is high for genes in the center of the diagram where histones are (no variation)
How many genes are the same in humans?
Those in the red circle of the diagram, ~99.9%
What is true of the genes in the outer circle of the diagram?
Low selection, greater variance. Gene isn’t so important and can tolerate some variation. Different allele frequency in different populations depending on advantage to be selected for, but aren’t necessary for being human. Ie. height, skin color, etc. which are based on multiple genes and allele variation.
What are the MCM6 variants?
=lactase enhancer
-mutations in the locus determine lactase expression in enterocytes will persist into adulthood or shut off in childhood (latter most common)
Natural variation vs natural selection
As selection changes, see changes in frequency of alleles
All genes mutate but natural selection brings most back to their original state
Natural variants can be due to cultural, environmental changes (lactose tolerance). Can provide a natural selective advantage (skin color, O2 sensing)
What is the DNA damage frequency in humans per cell (x 5x10^13 for the number of cells in a body) and the different repairs that occur per cell/day?
~1M/cell/day BER
~60/cell/day NER
~60/cell/day DSB repair: HR or NHEJ
~60k/replication cycle MMR
What are 4 DNA damage sensors?
- ATM
- CHEK2
- MDM2
- FA (Fanconi Anemia Complex) = ongoing DNA repair sensors
What is TP53?
A tumor suppressor protein that can initiate a cell cycle delay or apoptosis, depending on the amount of DNA damage
DNA damage sensors report to TP53
Can damage to repair pathways be related to disease?
Yes
-BER - PARP1 (inhibitors can be detrimental)
-NER - xeroderma pigmentosa
-DSB repair by HR - BRCA1 and BRCA2 (w/o them DSB repair doesn’t work) - hereditary breast and ovarian cancer
-MMR - HNPCC/Lynch Syndrome due to MLH1
How many errors does polymerase make per replication cycle?
~60k
leaving mismatches
When polymerase makes errors, what MMR repairs it?
- exonucleolytic proofreading correct ~99% -> ~600 errors remain
- MMR (other pathways) correct ~99% (~6 errors remain)
What kind of enzyme is necessary with NER?
a helicase to remove the damaged fragment
-then DNA polymerase fills the gap and ligase seals the nick
What is another name for the DNA polymerase eta?
XP-V gene
people w/o this gene cannot insert “AA” and have xeroderma pigmentosum and are very sensitive to UV damage (which often creates “TT” dimers, so AA is appropriate) -> cancer
What are MUTS homologs?
MSH2/6 recognize mismatches of various size
What are MUTL homologs?
creates signal to fix mismatch that MUTS found.
-downregulation of this gene -> Lynch syndrome
