genet 270 lec 13- new gen repair
What are the 3 broad categories of general DNA repair mechanisms?
Repair associated with replication, dark repair (NER + recombination repair), and the SOS repair system.
What 2 features define “general repair” pathways?
They repair many types of DNA damage and recognize distortions in DNA structure caused by improper base pairing.
What are the 3 steps of proofreading by DNA polymerase?
Recognition of helix distortion, excision via 3′→5′ exonuclease, and correction by reinserting the correct nucleotide.
What are the 2 forms of proofreading functions found in cells?
Proofreading can be part of the polymerase enzyme itself or performed by a separate accessory protein.
What are the 2 E. coli polymerases with proofreading roles, and what does each do?
Pol I (repair polymerase with proofreading built-in) and Pol III (replication polymerase; its proofreading depends on DnaQ/mutD subunit).
What phenotype do dnaQ/mutD mutants show (2 items)?
They lack 3′→5′ exonuclease activity and therefore show dramatically increased spontaneous mutation rates.
What 4 types of DNA errors are recognized by methyl-directed MMR?
Mismatches, frameshifts, base analog incorporation, and minor distortions from alkylation.
What is the key difference between mismatch repair and proofreading? (1 key distinction)
Proofreading occurs at the replication fork, while mismatch repair occurs after replication, scanning hemi-methylated DNA.
What enzyme methylates newly replicated DNA, and how does this enable strand discrimination? (2 steps)
Dam methylase methylates the parental strand first, leaving the daughter strand unmethylated, allowing the system to know which strand to repair.
What are the 6 ordered steps of MutS–MutL–MutH mismatch repair?
MutS binds mismatch
MutL + MutH join
DNA slides to a hemimethylated GATC
MutH cuts unmethylated daughter strand
Exonuclease + UvrD remove DNA past mismatch
Pol III + ligase resynthesize and seal the gap
What are the 2 major reasons mutS, mutL, and mutH are in the same pathway?
Mutations in these genes yield non-additive effects and were identified as mutator mutants in the same genetic screens.
In the λ heteroduplex experiment, what 2 observations proved MMR corrects mismatches?
Progeny phage always exhibit the dam⁺ parent phenotype and mismatches in hemimethylated DNA are corrected toward the methylated strand template.
What are the 3 reasons dam⁻ mutants are hypersensitive to 2-aminopurine?
DNA is not methylated, so MMR cannot pick the correct strand
The repair system cuts both strands at nearby AP sites
This produces lethal double-strand breaks
What are the 2 types of dark repair originally proposed?
Photoreactivation (light-dependent) and dark repair (light-independent); later shown to involve multiple pathways like NER and recombinational repair.
What is liquid-holding recovery and what does it show? (2 points)
UV-irradiated cells kept in non-nutrient buffer survive better later, showing light-independent repair processes operate even in phr⁻ mutants.
What type of damage does NER specifically recognize (1 major category)?
Major helix distortions, such as pyrimidine dimers or bulky adducts.
What are the 4 core Uvr proteins and their functions?
UvrA₂ = DNA damage scanning
UvrB = binds lesion and cuts 3′ side
UvrC = cuts 5′ side
UvrD = helicase that removes the damaged oligo
What are the 7 mechanistic steps of NER (UvrA–UvrB–UvrC–UvrD)?
UvrA₂–UvrB scan DNA
Lesion recognized
UvrA leaves, UvrC binds
UvrB cuts 4–5 nt 3′
UvrC cuts 7–8 nt 5′
UvrD removes fragment
Pol I + ligase fill and seal
What is the purpose of postreplication repair (1 core function)?
It allows replication to continue past unrepaired damage, leaving a gap that will be fixed later by recombination.
What are the 3 key events in RecA-mediated gap repair?
RecA binds ssDNA at the gap
Promotes strand invasion of sister duplex
DNA Pol III fills the gap using the healthy strand as template
According to the diagram on page 21, what happens when Pol III encounters a pyrimidine dimer? (2 steps)
Pol III stalls at the dimer
Replication restarts downstream using a new primer, leaving a single-stranded gap
What enzyme resolves the Holliday junctions formed during recombination repair?
RuvABC complex resolves the Holliday junctions and restores two double-stranded molecules.
What are the 3 triggers or characteristics indicating SOS activation?
Requires high DNA damage, produces error-prone repair, and increases survival by bypassing lesions.
What are the 4 steps of RecA activation leading to SOS induction?*
DNA damage produces ssDNA
ssDNA binds RecA, forming RecA*
RecA* stimulates autocleavage of LexA
SOS genes derepress, increasing repair protein expression
What 3 events does RecA* cause during SOS induction?
Cleavage of LexA (SOS derepression)
Cleavage of λ cI (lysogenic induction)
Activation of UmuD → UmuD′, necessary for mutagenic Pol V
What are the 2 domains of LexA and why do both matter?
LexA has a dimerization domain and a DNA-binding domain; cleavage separates them, preventing LexA from repressing SOS genes.
What 2 events convert UmuD into its active form?
RecA* stimulates cleavage of UmuD → UmuD′, which then pairs with UmuC to form Pol V.
What is the “relaxed clamping model” of Pol V?
Pol V replaces the β-clamp interaction of Pol III, allowing the polymerase to synthesize across damaged bases without pausing for proofreading.
What experimental result proves SOS mutagenesis requires RecA?
In the his⁻ reversion assay, UV mutagenesis occurs in wild-type, uvr⁻, and recBC⁻, but never in recA⁻ mutants, proving RecA is essential.
What did Weigle reactivation show (2 conclusions)?
Irradiated phage survive better on UV-irradiated hosts
More phage progeny become mutant, proving an inducible mutagenic repair pathway
Scenario: A dam⁻ mutant is exposed to 2-aminopurine (2-AP). What lethal event occurs and why?
The mismatch repair system cannot identify the daughter strand, so it cuts both strands near AP mismatches, creating double-strand breaks, which kill the cell.
A mutH⁻ mutant is exposed to mismatches after replication. What happens to mutation rate and why?
Mutation rate increases sharply because MutH is the endonuclease that cuts the unmethylated daughter strand, so mismatches cannot be removed.
A cell has normal MutS/MutL but UvrD helicase is deleted. What part of mismatch repair fails?
Excision fails because UvrD cannot unwind and remove the damaged fragment, so the mismatch remains.
A bacterium with a mutS⁻ mutation is exposed to a base analog (2-AP). Predict the outcome.
The cell will survive better than a dam⁻ strain, but show high mutation frequency, because mismatched bases are never recognized for repair.
A UV-damaged cell is kept in non-nutrient buffer for several hours (liquid-holding recovery). What increases and why?
Survival increases because dark-repair pathways (NER + recombination repair) operate while the cell is not dividing.
A strain is uvrA⁻ but recA⁺. What UV-sensitive phenotype do you expect?
Severe UV sensitivity because the cell cannot perform NER, the main pathway that removes pyrimidine dimers. Recombination repair works but cannot fully compensate.
A strain is recA⁻ but uvrA⁺. What happens after UV damage?
NER removes dimers, so survival is better than recA⁻ alone, but the cell cannot perform recombination repair or SOS induction, making it highly UV-sensitive and unable to tolerate replication-blocking lesions
DNA Pol III encounters a thymine dimer. What immediate event occurs and what is left behind?
Pol III stalls, then reinitiates downstream, leaving behind a single-stranded gap opposite the unrepaired dimer.
RecF pathway fails to load RecA onto the ssDNA gap. What part of recombination repair cannot occur?
Strand invasion cannot occur, so the gap opposite the lesion never receives a healthy template, blocking completion of postreplication repair.
A Holliday junction is formed during recombination repair but RuvABC is deleted. What happens?
The junction cannot be resolved, leaving chromosomes linked and non-segregatable, likely causing cell death.
A cell produces RecA* but has a lexA(Ind⁻) mutation (LexA cannot be cleaved). What happens to SOS induction?
SOS genes are not induced, because LexA is uncleavable, so even RecA* cannot derepress the SOS regulon.
A bacterium is exposed to high UV and lacks UmuDC. What happens to survival and mutation frequency?
Survival drops sharply and UV mutagenesis is absent, because Pol V (UmuD′₂C) is required for error-prone lesion bypass.
A λ lysogen is UV-irradiated in a recA⁻ host. Does phage induction occur? Why or why not?
No induction occurs because RecA* is required to cleave the λ cI repressor, so the prophage remains lysogenic.
A lexA⁻ null mutant (no LexA protein) experiences DNA damage. What happens to SOS gene expression?
SOS genes remain constitutively ON, because there is no LexA repressor, leading to continuous expression of din genes—even without damage.
A cell overexpresses UmuD and UmuC, but RecA is absent. Will Pol V be active?
No—UmuD cannot become UmuD′ without RecA*-mediated cleavage, so Pol V never forms, and error-prone repair will not occur.
A strain contains a point mutation in MutL that prevents interaction with MutH. What effect does this have on mismatch repair?
Mismatch repair fails because MutH cannot be recruited to the MutS–MutL complex, so the unmethylated daughter strand is never nicked.