Gene alteration and Repair Flashcards
What are adductomics?
The identification of DNA damage at the earliest stages of cancel development
What are DNA deviations associated with?
Destabilization of cellular metabolic homeostasis
What is the reason for most endogenous DNA damage?
DNA engaging in hydrolytic and oxidative reactions from water and Reactive oxygen species that are already present in the cells
How many single strand breaks are estimated to happen an hour due to ROS?
2300
What are the uncommon types of DNA lesions?
DNA deamination and methylation
What is special about base deamination?
It is a source of spontaneous mutagenesis and is exacerbated by single strandedness
When do Abasic (AP) sites form in DNA?
When the N-glycosyl (backbone) hydrolyzes
How many abasic sites are created a day?
10K
What is the problem to DNA with abasic sites?
They are inherently unstable and convert into SSBs easily
What is the mechanism of methylation?
S-adenosylmethionine is a methyl donor, and can cause 4000 N7 methylguanine, 600 N3 methyladenine, and 10-30O6 methylguanine residues per cell per day
What are the methylating agents?
S-adenosylmethionine and nitro sated bile salts
What is cytosine deamination?
It is cytosine spontaneously losing an amino group to yield uracil
What family of enzymes catalyzes cytosine deamination?
APOBECs
What are the good and bad effects of APOBECs?
They can inhibit retrovirus replications like HIV, but can lead to mutations and are found in a lot of tumors
What is the most human affecting APOBEC?
APOBEC3C
What are the upsides of high and low fidelity DNA replication?
High fidelity: maintaining genetic information over generations and avoiding bad mutations
Low fidelity: Evolution + diversity
What is the differing features of different DNA polymerases?
Their fidelities
What protein complex is responsible for DNA replication?
Replisome
What directions does DNA polymerase 3 go in?
Replication is 5 to 3
Repair is 3 to 5
What is special about the E. Coli DNA Pol 3?
It synthesizes DNA fast
What are the two steps of DNA synthesis?
Initiation and elongation
Describe the initiation process of DNA synthesis
Unwinding - helicase unwinds the helix
Primase - primase synthesizes a RNA primer to provide a starting point for DNA synthesis in the lagging strand
Describe the elongation process of DNA synthesis
Leading strand synthesis - synthesized in the 5 to 3 direction (3 to 5 strand is template)
Lagging strand synthesis - short segments (Okazaki fragments) put together by the primer - 5 to 3 strand is template
Primer removal and replacement - primers replaced with DNA nucleotides
Ligation - Ligase seals the gaps between Okazaki fragments
Which strands of the DNA does the leading and lagging strand of DNA follow in DNA synthesis?
Leading - uses 3 to 5 strand
Lagging - uses 5 to 3
What protein complex is responsible for the primer removal and replacement?
DNA polymerase 1
What is the Pol 3 core made of?
Alpha
Epsilon
Theta
What are the roles of the Pol 3 core subunits?
Alpha - DNA polymerase
Epsilon - 3 to 5 proofreading exonuclease
Theta - stabilizing factor
How can E. Coli Polymerase 3 have a higher mutation rate? What region of the complex is targeted?
A defect in the epsilon region
What is the starting event during a polymerase 3 error?
A misinsertion error in either DNA strand
What are the outcomes of a misinsertion error in E coli DNA?
It may extend the mismatch or yield a mutation or use the internal proofreading activity
How does extrinsic proofreading work?
Pol 3 may dissociate from the mismatch and polymerase 1 2 4 or 5 will activate
How many individuals were involved in the 1000 genomes project?
2500
How many genetic variants were found in each person in the 1000 genomes project?
88 million total, 35k per person
What are the carcinogens associated with paint?
Aromatic amines
What are the carcinogens associated with construction?
Silica and asbestos
What is the process called when molecules insert themselves between DNA base pairs?
DNA intercalation
What types of molecules are intercalators?
Planar aromatic molecules
What is the mechanism of alfatoxins?
They are not intercalators themselves, but instead their metabolites interact with DNA and intercalate
What is a DNA adduct?
It is a structural change in DNA resulting from the attachment of chemical compounds that is carcinogenic
What is the ethanol metabolism product that is carcinogenic?
Acetaldehyde
What group of acetaldehyde is dangerous? Why?
The carbonyl group is highly electrophilic, which reacts with guanine
What is the carcinogenic structure associated with adjacent deoxy guanine residues?
Intrastrand crosslinks
What are the differences in effect of DNA adduction and Intrastrand crosslinks?
Adduction - mutations
Crosslinks - DNA replication and transcription
Why is aldehyde genotoxic?
It can cause a variety of DNA lesions
What is the problem associated with GG adducts in the presence of acetaldehyde?
Acetaldehyde prevents restriction enzymes from digesting DNA fragments
The GG lesions are therefore resistant to digestion, and cannot be detected on agarose gel
What are the health risks associated with aflatoxins and where are they found?
It is a fungal parasite carcinogen that causes liver and other cancers
What restrictions are in place for aflatoxins in Canada and are they being met?
Canadian Food Inspection Agency has monitoring programs, but significant food samples exceed the regulatory limit (20ug/kg)
What dye was used in Lab 5?
Orcein dye
What are the two hypothesized mechanisms of orcein dye?
- pH interaction with negative groups
- Chromatin hydrophobic interaction
What are the 4 ways the body deals with DNA damage?
- Removal of DNA damage and the restoration of the DNA duplex
- Transcriptional response
- Activation of DNA checkpoint to arrest the cell cycle
- Apoptosis to eliminate damaged/deregulated cells
What are the three types of DNA damage that change the helix structure?
- Replication/recombination
- DNA base damages
- DNA backbone conformation changes
- Covalent cross links
What type of DNA damage is Abasic sites, and single and double strand breaks?
DNA backbone conformation changes
How are DNA conformational changes sensed?
sensor proteins slide along DNA to detect conformational changes
What types of proteins scan for MMR DNA mismatch repair? What do they detect?
MutS detect insertion/deletion and wrongly inserted bases
What is the three step process of MutS repair?
- MutS scans DNA
- Mismatched DNA creates a conformation change when going by a protein MSH6
- MSH2 closes to MSH6, stabilizing the DNA state
What are the levels of DNA repair?
Sensors -> Mediators -> Transducers -> Effectors
What is the role of a mediator?
It transfers the sensor signals to the transducer proteins
What is the role of a transducer protein?
It activates target proteins for effectors
What are the main effector compounds?
p53 and cdc25
What are the three proofreading eukaryotic processes?
- Nucleotide specificity - the correct dNTP is usually incorporated in the first place
- When the incorrect base is added, some DNA has 3 to 5 activity that can remove the mismatch during replication
- Extrinsic DNA mismatch repair
How does DNA polymerase combine polymerase and exonuclease activity?
- The polymerase monitors correct base pairing
- DNA polymerase 2 adds nucleotides at the 3 end and also removes incorrect nucleotides
- 3 to 5 exonuclease activity mediates incorrect nucleotides
How many errors are corrected by DNA proofreading?
90-99.9%
What are the 2 extrinsic DNA polymerase proofreading types?
- Dissociation of synthesizing polymerase at a mismatched base
- Engagement of extrinsic polymerase excises the mismatched base
Does extrinsic proofreading happen before or after the initial synthesizing polymerase pass?
After
What kinds of DNA damage are repaired by the exonuclease proofreader epsilon in polymerase 3?
Bulky lesions (crosslinks)
Double strand breaks
What proteins repair UV damage? What is the process called? Where is this process found?
Photolyase repairs the damage in a process called photoreactivation, it is found in many animals but not humans
What are the 5 steps of photoreactivation for thymine dimers?
- Binding - Photolyase binds
- Absorption of light - this activates the photolyase
- Electron Transfer - the photolyase transfers an electron and breaks the base bonds
- Restoration - Thymine bases are restored
- Dissociation - Photolyase dissociates from the DNA
What is a thymine dimer?
A UV induced type of damage for thymine where two adjacent ones bond together
What are the two thymines bonded together in UV damage called?
A cyclobutane pyridine (CPD)
How does the photolyase repair the thymine dimer?
It flips out the dimer and positions it in the active site
What does the presence of uracil indicate?
Cytosine’s loss of the amino group
What enzyme removes uracil from the backbone?
glycosylase
What enzymes are active in base excision repair?
Glycosylase for the removal of uracil, other enzymes for the removal of the nucleotide, polymerase to replace the cytosine, and DNA ligase to seal the strand
What type of DNA repair is associated with thymine dimers in humans?
Nucleotide - excision repair
What are the 4 steps of nucleotide-excision repair?
- The damage is found by exinuclease
- 12 Nucleotides are removed
- DNA polymerase fills the gap
- DNA ligase seals the strand
What type of repair is activated when there is a mismatch during cell division?
Mismatch mediated excision repair
What enzymes detect in mismatch mediated excision repair?
MutL and MutS and MutH
What are the 5 steps of mismatch mediated excision repair?
- The damage is detected
- MutH will recognize the original strand
- The faulty copy is cut
- The mismatch is removed from the new strand
- Polymerase fills the gap and ligase seals
What are the two types of double strand break repairs?
Homologous recombination and non-homologous end joining
What is the non-homologous end joining process?
- Recognition and binding
- End processing
- Ligase joining (Ligase 1 and 4)
What ligases are used for which types of DNA damage?
Single strand break - ligase 3
Bulky lesions crosslinks - Ligase 1
Double strand breaks and Base mismatches - Ligase 1 and 4
What is the homologous recombination process?
- The break is recognized, and the ends are processed with single strand overhangs
- The strands invade a homologous sequence and are cut according to the Holliday junction
What happens in an excess of DNA damage?
Apoptosis pathway!
What are the three potential outcomes for damaged cells?
They are repaired and returned to function, they undergo apoptosis if there is too much damage, or repair fails and damage accumulates and generates a tumor
