genet 270 lec 12- specific repair
What are the 2 broad sources of mutation in cells?
The 2 broad sources are mistakes during DNA replication and chemical/environmental factors (such as heat, chemicals, and irradiation).
What are the 2 key features of DNA replication fidelity and errors?
DNA replication usually has high fidelity, but occasional errors still occur, which can introduce mutations.
What are the 3 main types of chemical/environmental factors that cause DNA damage, and what does each do?
Heat causes deamination of bases; chemicals add groups to bases or sugars, break bonds, or fuse molecular parts; irradiation deposits photon energy in DNA, causing bond breakage or fusion of DNA parts.
What are the 2 main biological readouts used to detect DNA damage, and what do they measure?
Survival curves and mutant sensitivity are used; survival curves measure the fraction of surviving cells/plaques vs dose, while mutant sensitivity compares how well DNA repair–defective strains survive mutagen exposure.
In a survival curve of irradiated bacteria, what are the 2 key axes and what do they represent?
The y-axis shows percent survival (or fraction surviving), and the x-axis shows irradiation time or dose, often on a logarithmic scale
What are the 2 main observations from UV survival curves that indicate DNA damage?
As UV dose increases, survival decreases, and there is usually an initial flat region where early damage does not immediately kill cells.
What are the 2 main wavelengths relevant to UV damage vs protein damage, and which kills cells more effectively?
260 nm, where DNA absorbs strongly, kills cells much more effectively than 280 nm, where protein absorbs more strongly; thus DNA is the main target of lethal UV damage.
What are the 2 main pieces of evidence that chemicals/irradiation kill by damaging DNA?
First, UV at 260 nm kills cells more than 280 nm (implicating DNA); second, mutant strains defective in DNA repair are more sensitive to killing by mutagens.
What are the 2 key differences in survival curves between wild-type and DNA repair–defective mutants when exposed to mutagens?
DNA repair–defective mutants show a steeper drop in survival and reduced overall survival, indicating that their repair capacity is impaired compared with wild-type.
What are the 2 major mechanisms by which mutagens kill cells?
Mutagens kill cells primarily by causing blocks in DNA replication (e.g., stalled DNA polymerase) and double-strand breaks that cannot be repaired, leading to cell death.
What are the 2 key features of mutagens that block DNA replication (e.g., UV-induced pyrimidine dimers)?
These mutagens often do not affect base pairing directly but distort the DNA helix (as with pyrimidine dimers), causing DNA polymerase to stall during proofreading.
What are the 3 main steps in how pyrimidine dimers cause replication blocks?
UV forms pyrimidine dimers, the dimers remain correctly base-paired to the opposite strand, but DNA polymerase detects the distortion and stalls because it cannot correct the lesion.
Common exam mistake: Thinking dimers “break base pairing”; actually they distort structure, not the coding information itself.
What is the 1 key consequence of double-strand breaks in DNA?
Double-strand breaks are usually irreparable in bacteria, leading directly to cell death.
What are the 2 characteristic features of survival curves that indicate DNA repair is occurring?
Survival curves show an initial plateau at low doses (where repair is efficient and prevents death) followed by a steep decline at higher doses when repair systems become saturated or damaged.
What are the 2 main observations that define photoreactivation as evidence for DNA repair?
Irradiated bacteria survive better when exposed to visible light (300–600 nm) before plating, and this is due to activation of a photolyase enzyme that specifically cleaves thymine dimers.
In phage experiments, what are the 2 critical conditions that demonstrate photoreactivation?
UV-irradiated phages form more plaques on bacteria that were pre-exposed to visible light, indicating that host photolyase repairs phage DNA dimers during infection.
What are the 2 key features of DNA repair mutants that support the existence of repair pathways?
DNA repair mutants show diminished survival after mutagen treatment, and their mutations map to genes encoding DNA repair enzymes.
What are the 2 broad types of DNA repair pathways, and what do they recognize?
Specific repair pathways recognize particular types of lesions (e.g., deaminated bases), while general repair pathways recognize distortions in DNA caused by many kinds of damage.
What are the 5 specific repair targets covered in this lecture?
The 5 specific repair targets are deaminated bases, T:G mismatches from 5-methylcytosine (VSP repair), oxidative damage, alkylation damage, and pyrimidine dimers.
What are the 3 general DNA repair categories mentioned in this lecture?
The 3 general repair categories are CH₃-directed mismatch repair, nucleotide excision repair, and DNA damage tolerance/SOS inducible repair.
What are the 5 standard steps in the common base-excision–like repair theme?
(1) A damage-specific glycosylase removes the abnormal base; (2) an AP endonuclease cuts the backbone at the AP site (usually 5'); (3) the 5'→3' exonuclease activity of DNA Pol I degrades the damaged strand; (4) DNA Pol I fills in using the 3'-OH as a primer; (5) DNA ligase seals the nick.
What are the 2 key enzyme classes that initiate and process AP sites in this common theme?
DNA glycosylases initiate repair by cleaving the base from deoxyribose, and AP endonucleases cut the DNA backbone next to the AP site to allow replacement.
What are the 2 major roles of DNA Pol I in this common repair mechanism?
DNA Pol I provides 5'→3' exonuclease activity to remove damaged nucleotides and polymerase activity to synthesize new DNA using the intact strand as template.
What are the 2 main sources of base deamination, and what general type of mutation do they cause?
Deamination arises from mutagens (e.g., hydroxylamine, bisulfite, nitrous acid) and spontaneously, typically causing transition mutations such as GC→AT or CG→TA.
What are the 2 main chemical mutagens that convert cytosine to uracil and what mutation does this lead to?
Hydroxylamine (HA) and bisulfite both convert C→U, which, after replication, typically results in a GC→AT transition.
What 3 base changes can nitrous acid cause, and what general mutation patterns result?
Nitrous acid can deaminate C→U, A→hypoxanthine, and G→xanthine, leading to GC→AT and AT→GC transition mutations.
What are the 2 important spontaneous deaminations involving cytosine derivatives, and what are their consequences?
C→U and 5-methylcytosine (5-CH₃-C)→T occur spontaneously, causing GC→AT and CG→TA transitions, respectively, if not repaired.
Most likely long-answer angle: Why 5-methylcytosine is a hotspot for mutations.
What are the 4 main enzyme activities involved in repairing deaminated bases?
The repair uses damage-specific DNA glycosylases, AP endonuclease, the 5' exonuclease and polymerase activities of DNA Pol I, and DNA ligase to restore the correct base.
In 3 steps, how is a deaminated base (e.g., U in DNA) corrected?
A glycosylase removes U, AP endonuclease cuts the backbone, then DNA Pol I and ligase replace and seal the correct nucleotide.
What is 1 key conceptual reason why glycosylases are so important in deamination repair?
Glycosylases provide lesion specificity, recognizing and removing inappropriate bases (like U in DNA) while leaving the sugar–phosphate backbone intact for subsequent repair.
What are the 3 key features of 5-methylcytosine (5-CH₃-C) that make it a mutation hotspot?
5-methylcytosine is formed by Dcm methylase at the second C in 5'-CCWGG-3' / 3'-GGWCC-5', is used in restriction/modification and gene regulation, and its deamination produces T, creating a T:G mismatch hotspot.
What are the 2 main situations in which VSP repair is needed instead of standard mismatch repair?
VSP repair acts on T:G mismatches that arise from 5-methylcytosine deamination, especially when they occur after replication and methylation and thus escape standard mismatch repair.
What are the 3 main steps of VSP repair of a T:G mismatch?
VSP endonuclease binds the T:G mismatch and cuts next to T, DNA Pol I removes T and resynthesizes DNA, and DNA ligase seals the gap.
What are the 2 evolutionary clues suggesting VSP and Dcm are coevolved?
The VSP endonuclease gene lies next to the Dcm methyltransferase gene, and VSP repair specifically fixes the T:G mismatches produced by Dcm-methylated cytosines, indicating coevolution.
Application: In a 5'-CCAGG-3' site where 5-methylcytosine deaminates to T, what 2 alternative outcomes can occur if VSP repair fails?
The T can remain and pair with A, leading to a permanent transition mutation, or the G may be removed by other repair, potentially causing different local sequence changes depending on which strand is fixed.
What are the 3 main sources of reactive oxygen that damage DNA?
Reactive oxygen species arise from normal metabolism, environmental exposure, and chemical agents.
What is the 1 most common oxidative lesion in DNA and what base does it derive from?
The most common oxidative lesion is 7,8-dihydro-8-oxoguanine (8-oxoG), which is derived from guanine.
What are the 2 types of mutations that mispairing of 8-oxoG commonly causes?
8-oxoG mis-pairs with A, leading to GC→TA transversions, and related mispairing can also contribute to TA→GC transversions.
What are the 3 key bacterial proteins that limit mutations from oxidative damage and what is each one’s main role?
MutM (N-glycosylase/AP endonuclease) removes 8-oxoG from DNA, MutY (N-glycosylase) removes A mispaired with 8-oxoG, and MutT (phosphatase) degrades 8-oxodGTP to 8-oxodGMP, preventing its incorporation.
What are the 2 types of genes classified as “mut” genes, and what phenotype do they confer?
“mut” genes like mutM, mutY, mutT (oxidative repair) and mutD, mutS, mutL, mutH (proofreading and mismatch repair) are mutator genes whose mutation increases spontaneous mutation rates.
What are the 2 specific functions of mutD and mutS/mutL/mutH?
mutD (dnaQ) encodes the editing subunit of DNA Pol III, while mutS, mutL, mutH encode components of methyl-directed mismatch repair.
How do we know MutM, MutY, and MutT function independently (2 key observations)?
Mutations in each gene cause distinct increases in mutation frequency, and combining them produces additive increases, showing that they act in separate, nonredundant pathways.
Common exam mistake: Assuming they are all in one linear pathway; in fact they act independently at different stages (nucleotide pool vs DNA).
Scenario: If a bacterium lacks MutT but still has MutM and MutY, what are the 2 main consequences for DNA?
More 8-oxoGTP enters DNA, increasing 8-oxoG-containing bases, but MutM and MutY can still remove many lesions, partially limiting the mutation rate.
What are the 2 types of alkyl groups commonly added to DNA, and what are the 2 main structural effects?
Alkylating agents add methyl (CH₃) or ethyl (C₂H₅) groups to bases or phosphates, which can alter base pairing and cause subtle or large distortions in the DNA helix.
Which 2 kinds of repair pathways deal with major vs subtle helix distortions caused by alkylation?
Major distortions are handled by general repair pathways (e.g., nucleotide excision repair), while subtle distortions are corrected by specific alkylation repair pathways.
What are the 3 main strategies cells use for specific alkylation repair?
They use an adaptive response (Ada protein and inducible proteins), N-glycosylases such as AlkA, and direct reversal by methyltransferases that remove alkyl groups from specific positions.
What are the 3 key roles of the Ada protein in the adaptive alkylation response?
Ada acts as a methyltransferase that accepts alkyl groups, its N-terminal methylation converts it into a transcriptional activator that recruits RNA polymerase, and it induces expression of adaptive response proteins (including more Ada, AlkA, AlkB, AidB).
What is 1 key function of AlkA as an N-glycosylase in alkylation repair?
AlkA removes alkylated bases such as 3-methylguanine, 3-methyladenine, and 7-methylguanine, initiating base-excision repair of alkylated lesions.
How do O6-alkylguanine and O4-alkylthymine methyltransferases work (2 core steps)?
These methyltransferases transfer the alkyl group from O6 of G or O4 of T to a cysteine residue in themselves, which irreversibly inactivates the enzyme (“suicide” inactivation).
What are the 3 main sequential effects of N- and C-terminal methylation of Ada on the cell?
(1) N-terminal methylation changes Ada’s conformation to bind specific promoters and upregulate transcription/translation of Ada and glycosylases; (2) increased AlkA, AlkB, AidB expression removes more alkylated bases; (3) C-terminal methylation causes Ada to act as a direct methyltransferase on DNA and then be degraded, no longer activating transcription.
Why are alkylation repair enzymes considered “inducible” (2 key reasons)?
Their expression increases upon exposure to alkylating agents, and Ada’s N-terminal methylation directly converts it into a transcriptional activator, boosting the adaptive response.
Scenario: After a mild alkylating treatment, how does the adaptive response change the outcome of a subsequent stronger treatment (2 effects)?
The initial exposure induces Ada and related proteins, so during a later stronger treatment the cell has higher levels of repair enzymes, leading to increased survival compared with naïve cells.
What are the 2 main effects of UV irradiation on DNA bases?
UV irradiation causes bases to absorb energy strongly, increasing the reactivity of double bonds and resulting in abnormal covalent bonds that link adjacent bases, such as pyrimidine dimers.
What are the 2 key structural changes caused by pyrimidine dimers in DNA?
Pyrimidine dimers covalently link adjacent pyrimidines and create a kink or distortion in the DNA helix, interfering with replication and transcription.
What are the 3 main features of photoreactivation as a repair mechanism for pyrimidine dimers?
Photoreactivation specifically repairs cyclobutane pyrimidine dimers, was the first DNA repair system discovered, and uses visible light (300–600 nm)–activated photolyase to split fused bases.
What are the 3 historical/experimental facts about the discovery of photoreactivation?
In the 1940s, Albert Kelner showed that Streptomyces griseus survives UV better in light than in dark, leading to the discovery that photolyase repairs dimers using visible light.
What are the 3 main steps by which photolyase repairs a pyrimidine dimer?
Photolyase binds the fused bases, its FADH₂ cofactor absorbs visible light, and the absorbed energy drives separation of the covalently linked pyrimidines, restoring normal base pairing.
How do photoreactivation experiments with bacteria and phages together illustrate host–phage interaction in DNA repair (2 points)?
UV-irradiated phage DNA can be repaired by photolyase in the host bacteria, and plaque formation increases when infected bacteria have been exposed to visible light, showing repair before or during infection.
Concept check: What are the 2 differences between “damage” and “repair evidence” when looking at UV survival curves with vs without visible light?
Without visible light, UV causes steeper survival decline (damage only), whereas with visible light, curves shift upward (higher survival), directly indicating active repair via photoreactivation.
What are the 3 coordinated roles of MutM, MutY, and MutT in preventing 8-oxoG–induced mutations?
MutM removes 8-oxoG from DNA, MutY removes A paired with 8-oxoG, and MutT hydrolyzes 8-oxo-dGTP in the nucleotide pool so it cannot be incorporated. Together they stop both formation and propagation of GC→TA transversions.
What are the 4 mechanistic steps MutM uses to remove 8-oxoG from DNA?
MutM recognizes the lesion, glycosylase activity removes the damaged base, AP endonuclease activity cuts the backbone, and downstream Pol I + ligase fill and seal the corrected site.
Why does 8-oxoG cause GC→TA transversions (2 mechanistic reasons)?
8-oxoG forms anti–syn mispairs with A, and replication uses this mispair as a template, inserting T opposite the A, locking in a GC→TA mutation.
In VSP repair, what are the 3 steps the VSP endonuclease uses to correct T:G mismatches from 5-methylcytosine deamination?
VSP endonuclease recognizes the T:G mismatch in 5′-CCWGG-3′ sites, nicks the DNA near the T, and Pol I removes and replaces T, followed by ligase sealing the strand.
Why is 5-methylcytosine a mutation hotspot (2 conceptual reasons)?
5-methylcytosine spontaneously deaminates to thymine, creating a normal-appearing T that escapes mismatch repair, making mutation fixation more likely.
What are the 3 key functional transformations the Ada protein undergoes during the adaptive alkylation response?
Ada transfers alkyl groups to itself, N-terminal methylation activates Ada as a transcription factor, and C-terminal methylation inactivates it as it performs direct repair
How does Ada activate transcription of alkylation repair genes (3 mechanistic steps)?
Methylated Ada binds adaptive-response promoters, recruits RNA polymerase, and upregulates AlkA, AlkB, AidB, and more Ada, amplifying repair capacity.
What are the 2 main ways AlkA participates in base-excision repair of alkylated bases?
AlkA removes alkylated bases via glycosylase activity, creating an AP site, which is then processed by AP endonuclease → Pol I → ligase.
What are the 3 energy-dependent steps photolyase uses to repair pyrimidine dimers?
Photolyase binds the dimer, absorbs visible light using FADH₂, and uses that energy to split the covalent bond, restoring normal base pairing.
What are the 4 steps in the general base-excision repair mechanism shared by many specific repair pathways?
(1) A glycosylase removes the damaged base, (2) AP endonuclease cuts the backbone, (3) Pol I excises and resynthesizes the region, and (4) DNA ligase seals the final nick.