Quiz #3 (ch 12,13) Flashcards
transition vs transversions
transitions: pyrimidine replaces pyrimidine or purine replaces purine
transversions: purine and pyrimidine are interchanged
trinucleotide repeats
can expand due to template slippage WITHOUT causing frame shift
Huntington disease
autosomal dominant disease that causes progressive neurodegeneration (only expands in males)
CAG (Gln)
HTT gene
normal range: 11-35
threshold for disease: >35
fragile-X syndrome
1/1500 men
most common cause of inherited mental retardation
CGG repeat just upstream of FMR1 (protein involved in regulating translation)
CGG copies:
normal: 6-54
transmitting: 52-230 (full mutation to grandchildren)
affected: 230-2300 copies (inactivates FMR1)
this repeat only expands during meiosis in females
effect of a crossover within an inversion
- meitotic products are generally not viable
- viable meiotic products usually do not contain crossovers within the inverted region
- provides a way to keep a set of genes for an adaptive trait together (genes for multiple queens & aggressive behavior in fire ants are contained within an inversion relative to other ant species)
reciprocal translocations
- ex: Philadelphia chrom
- 95% of individuals with chronic myelogenous leukemia (CML) have this translocation
- normal ABL proto-oncogene is tightly regulated
- constitutively active BCR-ABL oncogenic fusion protein
Gleevec
- aka imatinib
- designed to inactivate the BCR-ABL fusion gene that drives most chronic myelogenous leukemia (kills cells but does not harm normal cells since normal cells do not have the chrom 9/22 translocation)
second generation drugs
in some patients cancer cells subsequently mutate to become resistant to Gleevec, so second gen drugs target resistant cells that have very similar mutations in the kinase domain of the BCR-ABL fusion protein
mutants are the …… of evolutionary change
engines
Guanine + Cytosine + EMS
O-6-Ethylguanine + thymine
GC –> AT transition
thymine + adenine + EMS
O-4-Ethylthymine guanine
TA –> CG transition
intercalating agents
proflavin, acridine orange, benzopyrene diol epoxide, EtBr
inhibit DNA replication, inhibit transcription, decrease fidelity of replication –> frame shifts
mutagens ……. DNA
chemically modify
UV light mutagens
pyrimidine dimers
50-100/ cell/ sec/ in sunlight
ionizing radiation
X-rays/gamma rays: high-energy high-frequency
make double stand breaks in DNA
causes of spontaneous mutations
- DNA polymerases insert incorrect nucleotides during DNA replication
- oxidative damage via by-products of normal cellular metabolism
- depurinatin and deamination
DNA polymerase mistake/ base analogs
- nts are in equilibrium bw standard (G=C, A=T) pairing and rare tautomeric forms that pair differently
- closed form orients substrates properly for catalysis (cannot completely clsoe with wrong dNTP)
- after base selection and proofreading, mistakes occur every 10^6 or 10^8 incorporation events (every generation)
- more tautomeric shift –> higher mutation rate
oxidative damage definition and most common product
- by-products of normal cellular metabolism or exposure to high-energy radiation create by-products of reactive oxygen species
- most common product: 8-oxo-G from Guanine (can bp with C or A; associated with many human cancers)
how many purines fall in a typical human cell?
10,000 per day
how many cytosines deaminate in a typical human cell?
100-500 per day
hydrolytic damage
attacks glycosydic bond
attacks C to turn to U (U can bp with A durin DNA replication)
DNA is the only molecule that is………
repaired
DNA lesions /day in E.Coli and Eukaryotes
E. Coli: 1K per day
Eukaryotes: 100k per day
what does Dam methylase recognize?
GATC
mismatch repair in E. Coli
- DNA pol misincorporates a nucleotide, creating a mismatch. The newly synthesized GATC site is hemimethylated
- MutS binds the mismatch and forms complex with MutL
- MutS-MutL scan DNA bidirectionally, forming a loop (requires ATP)
- MutS-MutL finds the nearest GATC site and recruits MutH, which cleaves newly synthesized unmethylated GATC sequence
- helicase 2 and pol 1 exonuclease unwind and degrade the newly replicated DNA strand past the mismatch
- pol 3 fills the gap
- ligase seals the DNA
what type of enzyme is MutH
nickase
why use pol 3 in E. coli mismatch repair?
distance between mismatch and Dam site can be >1KB
mismatch repair is expensive
error rate 10^6 - 10^8 –>10^10
Mismatch repair recognition step in eukaryotes
MutS homologs (MSH2/6) and MutL-like proteins (MLH2/PMS2) recognize alteration in the DNA backbone due to the mismatch created distortion
Mismatch repair repair steps in eukaryotes
- MSHs can interact with PCNA (sliding clamp), which is left on the lagging strand after replication and helps guide MSH to the nascent DNA strand for repair
- no hemi-methylated DNA. Nicks on the newly replicated lagging strand (from Okazaki fragments) are recognized, and exonucleases cleave from nick to site of damage)
mutations in mismatch repair enzymes increase
inherited cancer susceptibility
ex: nonpolyposis colorectal cancer = 15% of colon cancers )genetic form of cancer)
methyltransferace
- can directly reverse O6-methylguanine
- can only do it once and then it’s dead
- more expensive because the enzyme becomes irreversibly modified and can only perform one reaction
BER general step
- abasic sites recognized by AP endonuclease
- backbone is cleaved and a short segment is resynthesised
bacteria: resynth by pol 1
euk: pol 1 extends from 3’ end and flap endonuclease makes a flap and cleaves displaced strand (or pol B can add 1 nt) - ligase seals gap
Nucleotide excision repair (NER)
can remove BULKY lesions
in e. coli
1. UvrAB scans dsDNA for distortions, melts short DNA region
2. UvrA is released
3. UvrC exinuclease is recruited and nicks on both sides of distortion
4. UvrD helicase removes fragment (10-13 nt)
5. repair with pol1 and ligase
Xeroderma pigmentosa
- cells/patients extremely sensitive to UV light
- UV light causes pyrimidine dimers and oxidative damage
- HUMANS DO NOT HAVE PHOTOLYASE, SO NER IS MAJOR REPAIR PATHWAY —>we rely on nucleotide repairs
- inability to repair DNA lesions leads to cancers like melanoma
- 40% die before age 40
stalling of RNA polymerase
damage –> stalled RNA pol recruits NER proteins –> transcription- coupled repair (ensures actively transcribed strands are repaired quickly)
cisplatin
forms cross links with N7 of 2 purines, in either same or opposite strand of DNA
makes it hard to fix cells –> apoptosis –> stops cancer cells from growing
horrible side effects
what happens when DNA pol encounters unrepaired damage?
blocks replication fork and RNA pol –> apoptosis
what can replication of unrepaired DNA or ds breaks cause?
replication fork collapse
what can help if pol 3 is stalled?
one of the translesion pol can replace it on the beta-sliding clamp
tanslesion (bypass) synthesis
pol 3 takes over after lesion has been passed
XPV
PolH bypass UV lessions
error free repair for thymidine dimers
involved in Xeroderma
mutagenic vs non-mutagenic translesion synthesis
mutagenic: E coli pol 4 & 5 and most translesion enzymes in euk insert “random nucleotides”
nonmutagenic: Rad 30 and human XPV only interact with thymidine dimers (cause insertion of TpT in complementary stand –> “error free” lesion bypass activity ( if dont have it, xeropigment cancer)
base excision repair (BER) specific step
specific glycosylases flip out DNA bases one by one to check for damage
uracil DNA glycosylase: deaminated C –> U
8-oxo-G DNA glycosylase
leaves an abasic (empty)(apurinic) site - doesnt have purimidine
how can the ends of a DS break rejoin?
homologous recomb (sister chromatid, non-sister, exogenous DNA)
nonhomologous end joining
DS break repair
- chew back ends with RecCBD until hit chi sequence (5’ to 3’)
- creates SS overhang with free 3’ OH
- code with RecA, which looks for homoloy
- displaces- invade sister strand making a D loop
- at this point have repaired but still intertwined with sister
. with recombinase, SS overhand ????
RecBCD
RecD helicase: unwinds 5’ to 3’
RecB helicase: unwinds 3’ to 5’
RecB nuclease: degrades unwound DNA (both strands)
RecC: recognizes chi sites scattered across the genome (decreased degradation of 3’ end; increased degradation of 5’ end)
RecBCD: processes 5’ ends and produces ss DNA with 3’ OH
RecA
- looks for homology; loaded onto 3’ extensions
- DNA-dependent ATPase (uses energy)
- slow nucleation because ssDNA is initially bound by ssB
- recruited by RecB
- coats ssDNA forming a RecA filament
Synthesis-Dependent Strand Annealing (SDSA)
run everything backwards; no recombination
1. strand dissociation and annealing (helicase to melt comp regions)
2. complete replication and ligate (DNA pol and DNA ligase)
holiday junctions
- 4-branched crossover junctions
- branch points can slide back and forth at no energetic cost
- cut same way: noncrossover
- differently: crossover and recombination (RuvAB: UV, sliding, cutting)(RuvC: nuclease)
RuvAB complex
repair of UV damage
reversible process but requires energy
moves 1000s of bases (far away) from cut
make a lot of heteroduplex DNA
stimulate branch migration and resolution (cleavage) of holiday junctions
RuvA: forms complex with 2 RuvB hexamers
RuvB: an ATPase that uses ATP from hydrolysis to drive branch migration
RuvC
nuclease recruited to RuvAB-Holliday junction and cleaves DNA (can be 1000s bp away)
Resolves holiday junctions in E. coli (does the actual cut)
what induces E. coli DNA repair (SOS) genes?
DNA-bound RecA (can act as a protease –> induce it and cuts itself)
what does LexA do?
ensures SOS genes are repressed in E. coli
E. coli SOS signal
- RecA binds ss (damaged) DNA and makes LexA cleave itself (RecBC helps bind it); cleaves lambda receptor
- lack of functional LexA allows SOS genes to be expressed
what is a key aspect of recmobination during meiosis 1?
double stranded break repair
how do you get the precise alignment needed for crossing over if you’re off by one base?
frame shift
ds homologous recombinatoin in meiosis 1
- Spo11: meisosis-specific endonuclease that attaches to DNA via tyrosine and makes ds cut
- Mre11-Rad50 removes Spo11-DNA, giving 3’ extensions
- extend 3’ ends, bind RecA, do normal DS repair (holliday junct)
- resolvases cleave DS holliday intermediate
make heteroduplex DNA
Sgs1 removes more DNA from 5’ end
hot spots of recombination
approx 30,000 spaced every 50-100kb (random-ish)
somatic recombination
- usually nonsister/homolog chromatids do NOT pair during mitosis and thus do NOT undergo crossover and recomb
- but as a consequence of DNA repair mechanisms, nonsister cromatid recombination
mitotic recomb wth crossover
mitosis of cell with one inactive copy of Retinoblastoma gene (heterozygous)
- normal: both daughters are heterozygous
- somatic: both daugher cells have
*
2 Rb- = cancer
consequences of branch migration
depend on source of homologous DNA
sister chromatids: should be identical except for rare mutations during replication
non-sister chromatids: alelic differences (mismatches in heteroduplex; repair; gene conversion)
gene converstions between loci that are similar but not identical
normally a good thing but soe weird things can happen
you can CREATE mutations
associated with diseases
recombinations used to repair stalled/damaged DNA replication forks
translesion synthesis: adds a few things to fix later; pol does not have proofreading
repair initiated: run over it
fork stalls: backward branch migration
lesion bypass
- replicate DNAP hops over lesion and continues
- more frequent on laggins trand because weird fragments are already being made anyway
- gap repair later
what happens if DNA pol encounters a SS region?
- fork collapses
- can repair by DS break repair (RecBCD, etc)
fork stalling
- DNA pol encounters lesion in leading strand, replication strand stalls and leads to backward branch migration
- fork regression: templates zipper back past lesion
- daughter strands come together
- gives a change for MMR, NER, BER systems to repair the lesion
- if not repaired, regressed fork looks like a DSB and can be a substrate for DSB repair pathway
2 options after fork regression
- lesion repair, digestion by nuclease, replication restart
- replication, branch migration, replication restart (take new strands and push forward past the lesion)
in mismatch repair ( ….. and ……. ) the other strand can be used as ……….
BER and NER
template
exogenous DNA ds break rejoin
- Cas9 can make a DS break anywhere in any organism
- can replace one allele with another (if you add homologous DNA, the Cas9 DS cut can be repaired via homologous recomb)
Chinese use of CRISPR
- co-receptor for HIV
- people who naturally don’t express CCR5 are resistant to infection by HIV, cholera, and chicken pox
*
nonhomologous ends joining
- Ku70-Ku80 bind to ends of ds break
- DNA-PKcs and artemis add phosphate to each other
- widening of ds break
- annealing (artemis nuclease chews back about 10 bases )
- XRCC5, XLF, DNA ligase 4 seal nicks (not very selective)
- ligation
good way to inactivate a gene
cipro
gyrase inhibitor
what happens when you stress a cell (give antabiotics)?
induce mismatch repair –> antibiotic resistance
sos response
error-prone DNA pol –> increase mutation rate
sister vs nonsister chromatid recomb
sister: S-phase, heteroduplex DNA, error-free repair
nonsister: NOT error free repair because allelic difference, mess up Mendelian ratios
