repair and recombination Flashcards
define mutation
permanent change in DNA sequence
T or F: most mutations are neutral or harmful
true
T or F: many mutations are advantageous
false; they’re rarely advantageous
list 2 ways in which mutations can be caused by
spontaneous mistakes in DNA replication or by environmental mutagens
what is a silent mutation
a mutation within non-coding DNA or a mutation that doesn’t affect the resulting protein product
what is the purpose of the ames test
to identify chemicals that promote mutations
define carcinogen
a chemical that promotes mutations
describe how the ames test works
use a bacteria that has been mutated to not produce His. Grow on a His free medium + put the mutagen in the middle. if the mutagen enhances mutations, the bacteria will be mutated to produce His, and we will see growth
T or F: different bacterial strains can be sensitive to different types of mutations
true
describe how rat liver extract can be used to predict if the mutagen would harm mammalian DNA as well as bacterial DNA
isolate extract that is His-. Plate on medium lacking His and incubate. Pos result = high number of His- to His+ suggests presence of a strong mutagen
what is mismatch repair
corrects non-complimentary mismatches made by DNA pol III that were missed by pol III proofreading
what is base-excision repair
removes bases with structural abnormalities
what is nucleotide-excision repair
removes the entire nucleotide when there is an abnormal base that causes distortions in the overall helical structure
what is direct repair
fixes damage or abnormal bases without removing the nucleotide first
dam methylation is used in which type of repair
mismatched repair
describe the activities of dam methylation
it permits discrimination of the template strand. It methylates both strands, but the template is methylated first so there is a short period in which it’s the only one that’s methylated. This is how we know which base pair in the mismatch was the correct one
during mismatch repair, what happens if both strands had become methylated
no repair occurs
during mismatch repair, what happens if neither strand had become methylated
repair occurs indiscriminately on broth strands
mismatch repair: which protein complex bind mismatched base pairs
MutL-MutS
structure of MutL-MutS?
clamp-like
T or F: MutL-MutS binds to mismatched base pairs in an ATP-dependent process
true
describe the mechanism of MutL-MutS
they bind to the mismatch and then slide along the strand until they reach a hemimethylated site
how far away does the mismatch need to be from the hemimethylated site
1000bp
which sequence is methylated by dam methylase
5’-GATC-3’
in the 5’-GATC-3’ sequences, which base is methylated + where on that base
adenine is methylated on the N6 position
what happens when MutL-MutS reaches the hemimethylated site
they encounter MutH
what does MutH do once MutL-MutS arrives at the hemimethylated site
MutH has endonuclease activity and will cleave the unmethylated strand on the 5’ side of the G in the GATC sequence
mismatch repair: describe what happens when the mismatch is on the 5’ side of the cleavage site
unmethylated strand in unwound and degraded 3’-5’ from nick to mismatch
mismatch repair: describe what happens when the mismatch is on the 3’ side of the cleavage site
unmethylated strand in unwound and degraded 5’-3’ from nick to mismatch
for mismatch repair, list 5 enzymes/proteins that are required
helicase, exonuclease, pol III, SSBs, and ligase
T or F: mismatch repair is very costly
true
why is mismatch repair costly
up to 1000 additional dNTPs are required to fix just one mismatched pair
in base-excision repair, what type of reaction causes the mistake
deamination
what is a common deamination
C to U
T or F: deamination is spontaneous + non-enzymatic
true
base-excision repair: which enzyme family recognizes base lesions
DNA glycosylases
what do DNA glycosylases do
they recognize base lesions and remove the offending base
how do DNA glycosylases remove the offending base in base-excision repair
they cleave the N-glycosyl bond
cleavage of the N-glycosyl bond leads to which type of nucleotide
abasic (lacking a base)
list the two types of abasic nucleotides
apurinic or apyrimidinic
in base-excision repair, how is it fixed if there is no glycosylase
the N-b-glycosyl bond between the pentose and the base can hydrolyze
base-excision repair: once an abasic nucleotide is present, what happens
AP (abasic) endonuclease nicks the DNA backbone either directly 5’ or directly 3’ to the missing base
base-excision repair: once an AP endonuclease nicks the DNA backbone beside the missing base, what happens
pol I uses its 5’-3’ exonuclease activity to remove a few bases starting from the nick + it replaces them. ligase then seals the nick
nucleotide-excision repair: what can cause large distortions in helical structure
UV light
nucleotide-excision repair: give an example of a large distortion to helical structure that can be caused by UV light
thymine dimerization
nucleotide-excision repair: describe how much DNA must be removed to make the repair
larger chunks need to be removed
nucleotide-excision repair: which enzyme is used to make the large repair
excinuclease
nucleotide-excision repair: describe the actions of excinuclease
hydrolyzes two phosphodiester bonds. One nick is created on either side of the lesion
nucleotide-excision repair: how many phosphodiester bonds does excinuclease hydrolyze
2
nucleotide-excision repair: once two nicks are made by excinuclease activity, what happens
helicase removes the offending DNA fragment, then pol I replaces them, and ligase seals the nick
which disease is caused by defects in nucleotide-excision repair machinery
Xeroderma Pigmentosum
describe the symptoms of Xerodoma Pigmentosum
extreme sunlight sensitivity, freckling, increased risk of skin cancers, irritated eyes/impaired vision, increased risk of eye cancers
name the enzyme that is very good at repairing thymine dimers
photolyase
which organisms have photolyase
bacteria and plants
T or F: humans synthesize photolyase
false! only plants and bacteria do
when is direct repair used (ie what was the damaged caused by)
direct repair is used to repair damage caused by alkylation
direct repair: give an example of alkylation that occurs
guanine –> O6-methylguanune
direct repair: what does O6-methylguanine base pair with
pairs with T instead of C
direct repair: describe the mechanism
methyltransferase can transfer the methyl of O6-methylguanine to one of it’s cysteines. This inactivates the protein permanently but regenerates the guanine
when do double stranded breaks and ssDNA occur
occur when a replication fork runs into already damaged DNA
what happens when the replication fork runs into already damaged DNA
the replisome usually falls off
how do double stranded breaks get repaired
by a mechanism involving homologous recombination
describe how homologous recombination is involved with fixing DNA with no template
the accurate nucleotides are determined from the homologous chromosome
DNA damage with no template: which enzyme is used to replicate DNA with lesions
DNA pol V
describe the error rate of DNA pol V
it can replicate over lesions that would normally stall other polymerases, but with a much higher error rate
what is homologous recombination
rearrangement of genetic info between chromosomes that share extended regions of (almost) identical sequences
homologous recombination: describe the events that occur after a replication fork collapses upon encountering a DNA nick and the 5’ end of the dsDNA is degraded
the exposed 3’ end is bound to recombinase, which facilitates another 3’-5’ strand to invade and undergo complimentary base pairing with the now exposed 3’ end. The structure can migrate along the strand and create an X-like crossover known as a Holliday intermediate. Nucleases cleave the Holliday, and the strands are re-ligated, and the replication fork is re-established
homologous recombination: what protein is responsible for the initial 5’ degradation
RecBCD
homologous recombination: which types of activity does RecBCD have
both helicase and exonuclease
homologous recombination: which is the recombinase that promotes strand invasion
RecA
homologous recombination: what does RecA do
it forms a long filament on ssDNA with up to several thousand subunits
homologous recombination: what does RecBCD do
assists in removing the SSBs from ssDNA and then allowing RecA to bind
list the 3 purposed of homologous recombination
helps repair DNA damage, helps to physically link chromatids for proper division during meiosis I, enhances genetic diversity
what does meiosis I separate
homologous chromosomes
T or F: cohesins keep homologues together
false! there are no cohesins. only sister chromatids have cohesins
what is aneuploidy
incorrect number of chromosomes in a daughter cell
in humans, when does egg production begin
week 12 of gestation (before female is even born)
describe meiosis from gestation to ovulation
meiosis is initiated in the fetal germ line cells. Cells proceed through most of meiosis I, where chromosomes line up and generate cross overs. The process then stops with the crossovers in place, until just before ovulation
T or F: the longer the arrested stage of meiosis, the higher risk of aneuploidy when the homologues finally separate
true
