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49 Cards in this Set
- Front
- Back
What's a transition mutation |
Purine to purine mutation |
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What's a transversion mutation |
pyrimidine to purine or vise versa |
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What's EMS damage |
EMS stands for ethylmethyl sulfonate - adds ethyl group to the free O6 of guanine residues (creating O6-ethylguanine), which causes a transition base pair to T instead of C (GC->AT) basically its the alkylation of guanine |
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Name two types of direct repairs and explain them |
Photolyase- enzyme that uses light that breaks covalent bonds (such as thymine dimers) O6-methylguanine-methyltransferase (dealkylation by alkyltransferase)- Asulfhydryl group of the enzyme accepts the methyl group(blue) from a guanine on the DNA, thus inactivating theenzyme. |
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What type of repair is DNA glycosylase? Describe it. |
Base Excision Repair, removes the damaged base, leaving apyrimidinic/apurinic site (AP site), Then AP endonuclease excises the site, DNA polymerase fills in thegap and the DNA strand issealed by DNA ligase. |
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Describe AP endonuclease |
APE-1 cleaves 5'of apyrimidinic site, DNA polymerase beta-fills gap, removes sugar, DNA ligase seals the nick, mistakes by DNA pol beta fixed by APE1 3'-exonuclease activity |
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What are the two steps to Nucleotide excision repair |
Incision - endonuclease cleaves both sides of damaged base (UvrABC excinuclease) Synthesis- replace with new base by polymerase/ligase seals nick |
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Explain NER in E.coli |
UvrAB - hydrolysis of ATP promotes uvrA+B dimerization, the complex scans DNA to find distortions, UvrA dissociates, UvrB melts DNA locally around distortion UvrC-forms complex with UvrB to nick around the damage UvrD (helicase) unwinds region to release the nicked single strand Pol1and ligase- repair/seal |
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Explain NER in humans |
XPC-hHR23B- complex finds damage XPA- recruits NER factors XPB,XPD - helicase subunits of TFIIH complex, unwinds DNA XPF, XPG - exonucleases that cut out damage strand RPA- positions XPF and XPG DNA pol - fills gap, ligase seals |
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Describe Mismatch repair in E.coli (Mut family of enzymes) |
MutS- dimer, recognizes mismatch because DNA backbone is distorted/kinked, requires ATPase of MutS to hydrolyze ATP, MutS recruits MutL and MutH MutH- endonuclease, cuts near mismatch Exonuclease digests strand with mismatch Repaired by Pol III and ligase |
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Describe how double-stranded break repair happens (NHEIJ non-homologous end joining) |
Ku70+Ku80 heterodimer - binds broken DNA ends, recruits: DNA-PKcs (DNA-dependent protein kinase, catalytic subunit), Artemis endo/exo nuclease activity to process broken ends Ligase IV, XRCC4, Cerunos-XLF to join broken ends |
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3 classes of Genetic recombination |
1. generalized/homologous recombination 2. Site-specific recombination 3. Transposition of mobile elements |
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Describe the RecBCD pathway in E.coli |
RecBCD- binds double stranded breaks -helicases (RecB (fast) RecD (slow) unwinds to a "chi site") Rec A - loads on 3'ssDNA after unwinding, helps invading strand find homologous region (forming D loop) Gaps are sealed, repaired, forms Holiday Junction RuvA (helicase), RuvB(ATPase) - enables branch migration RuvC-nicks 4-stranded junction to resolve |
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What are homologous in eukaryotes and E.coli in terms of the RecBCD pathway? What induces double stranded break during meiosis? |
Homologs of Rec (RecBCD, RecA, RuvA-B-Chomologs) and Ruv proteins Spo11, MRX complex - induces double stranded breaks during meiosis |
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What is RecA's participation presynapsis? How/what protein aids in this? |
RecA coats ssDNA 5'->3'' where each addition of RecA accelerates addition of more RecA towards the 3'end, aided by SSB which prevents RecA from trapping any ssDNA secondary structures that would hinder it from strand exchange |
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What is RecA's participation in synapsis? |
Alignment of complementary sequencesin ss and ds DNAs , alignment unlikely to be Watson-crick pairing due to low melting temp |
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What is RecA's participation postsynapsis? What experiment showed this? |
strand exchange, D-loop formation. Lehman: incubated 3H-labeled duplexP22 phage DNA withunlabeled P22 ss DNA in thepresence or absence of RecA - removed protein, filteredmixture throughnitrocellulose filter where D loops forming caused it to stick to the nitrocellulose filter |
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Two types of DNA damage |
structural distortions or single base pair |
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name single base pair change examples |
2 types of deanimation: cystine to uracil and adenine to hypoxanthine (created by guanine deaminase) and base pair change caused by errors in replication |
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Do single base pair damage disrupt transcription? Which ones do? |
no, but its damaging to future generations, structural do disrupt transcription and replication |
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How are pyrimidine dimers formed? |
Through structural base pair damage, by UV radiation, causing covalent linking to occur |
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Name 3 types of structural damages |
pyrimidine dimerization by UV, depurination (missing bases), and base alkylation (addition of bulky groups to products) |
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Electron rich centres in DNA are hot spots for what type of structural damage? why? |
alkylation, because the electrophile (wants electrons) so it gets it by attacking the phosphodiester bond |
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Who won the Nobel Prize for DNA repair mechanisms? |
Tomas Lindahl, Paul Modrich, Aziz Sancar |
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What is Direct Repair? |
damaged base is not removed, it is repaired on site |
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Describe the Human BER pathway |
glycosylase removes the mistake, APE1 cleaves apyrimidic side, DNA pol Beta fills in gap with correct bp and DNA ligase seals the nick. if DNA pol Beta makes a mistake, process is found with APE1's 3'-exonuclease activity |
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What is XP? and what is it caused by? |
Xerodermapigmentosum: UVexposure leads to skincancer XP is caused byexcision repair defects– XP human genes aresimilar to yeast RADexcision repair pathway |
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How does XP disorder occur? |
XP disorder is when the XP mutation does not appear in the XP gene, instead its DNA pol n (symbol is eta), where DNA pol n can replicate past T-T dimers, called DNA damage by pass |
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how does the repair system“know” which base to remove on which strand? |
THE MISMATCH REPAIR SYSTEM IDENTIFIESORIGINAL METHYLATED STRAND (because the newlysynthesized strand is not yet methylated) |
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what is a retrieval system? what protein does it require? |
they carry out post-replication/recombination repair, Gap is filled by “retrieving” the correspondingsequence from the normal duplex viahomologous recombination, requires recA |
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what is the SOS response? how is it activated in E.coli? |
last resort in DNA repair mechanisms, SOS response in E. coliis activated by DNAdamage or replicationblock: UV, alkyl, dnagene mutants • Induces transcription oflong-patch excision &recombination repairproteins (SOS responsegenes) • Activated RecA causesLexA repressorautoproteolysis • PolV is mutagenic... • Allows for replicationacross from the DNAlesion but often insertsthe wrong bases |
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2 ways double strand breakage can be repaired? |
1. Similar to bacterial retrieval repair involving homologousrecombination (S & G2) 2. Non-homologous end joining (NJEJ) in G1 |
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what binds to a NITROCELLULOSE FILTER BINDING ASSAY? |
ssDNA binds to the nitrocellulose filter, dsDNA does not,but if dsDNA is bound to the protein, it binds |
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How does a GEL MOBILITY ASSAY detect an interaction between a protein and DNA? |
detects an interaction between a protein and DNA by the reduction of theelectrophoretic mobility of a small DNA that occurs on binding to a protein (otherwise called a supershift) |
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What is the process of DMS footprinting? |
Treat DNA-protein complex with dimethylsulfate (DMS) tomethylate under mild conditions, so that only onemethylation event occurs per DNA molecule. Next, theprotein is dislodged, and the DNA is treated with piperidine,which removes methylated purines, creating apurinic sites,then breaks the DNA at these apurinic sites. |
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What is CHROMATIN IMMUNOPRECIPITATION(ChIP)? How does it work? |
First Isolate chromatin from cells, treat with formaldehyde (to form covalent bonds between DNA and any protein bound to it). Shear/break chromatin by sonication to produce short, dsDNA fragments cross-linked to proteins, IP with Abs directed against the protein of interest. Detects a specific protein-DNAinteraction in chromatin in vivo, usingan antibody to precipitate a particularprotein in a complex with DNA, andPCR to determine whether the proteinbinds near a particular gene. |
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What is YEAST TWO-HYBRID SYSTEM? How does it work? |
Assays for binding between twoproteins while utilizing DNA-binding andactivating domains This technique takes advantage of thestandard model of transcriptionactivation:1.) DNA-binding domain (BD) binding toan enhancer (pink) 2.) the activating domain (AD) thatinteracts with the basal transcriptioncomplex, recruiting it to the promoter. c) could use a cDNA library to screenfor interacting proteins by transformingyeast with different plasmids, but theassay is indirect, so should alwaysverify by a direct assay, such as IP. |
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How can you tell the difference between splice (crossover) products and patch (noncrossover) products? (homologous recomb) |
spliced is if the resolution of the holiday junction are not the same color, its patched if they are |
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Does RecBCD nick at chi sites to initiate recombination? what was the experiment? |
Yes, They 3’-end-labeled the DNA end with[32P]. Then, they incubated this end-labeled DNA fragmentwith (+) or without Chi (-) site with (+) or without (-)RecBCD for 30 sec gel shows that RecBCD cuts at chi sites and unwinds beyond the nick |
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What is a recombination hot spot? Can they function in both directions? |
Hotspot is Chi sequences that promote recombination/crossover No, Chi sequences are non-symmetric (which is why theyfunction only in one orientation): |
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Chi sites have an effect on what directly? |
recombination frequency |
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What is RecBCD's role? |
RecBCD helicase/nucleaseprocesses broken DNA moleculesto generate regions of ssDNArequired by RecA to initiate strandexchange |
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What's the difference between RecB and RecD? What is the result of RecB's movement? |
RecB-helicase that is slow moving on the 3' ending strand RecD-helicase that is fast moving on the 5' ending strand Slow RecBaccumulates a single-stranded DNA loop in the lower strandduring unwinding. After the enzyme encounters the Chi site,this loop is “reeled in” and its 3’ end, now containing Chi, isavailable for RecA assembly. |
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RecC does what? |
Recognizes Chi sites, Cleaves specific DNA strands at the holliday junction to finish recomb. |
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Where does RuvA bind? RuvB? What does RuvB do? Experiment that proved this? |
RuvA binds at the holiday junction, RuvB binds on both sides of RuvA, Ruv B functions as a motor that pumps DNA through the junction Parson and West (1993) constructed a labeled syntheticHolliday junction and mixed it with varying amounts ofRuvA and RuvB, treated with glutaraldehyde tocross-link proteins in the same complex and prevent theirdissociation, then gel mobility assay |
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In eukaryotes, programmed generation of DS DNA breaks occurs when and mediated by? |
programmed generation of DSB during meiosisis mediated by Spo11 and the MRX complex |
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In eukaryotes, what is the homologous recomb pathway? |
DSB formed by Spo11 and Mrx complex, 5' to 3' resection of the 5' ending strands at the break site, Strand exchange proteins Dmc1 and Rad51 then assemble on the ssDNA tails, strand invasion occurs then recombination |
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Mutations in what are thought to be responsible for half familial breast cancers? |
BRCA2 |
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What does Rad51 do? What is it a homolog of? What happens when it interacts with BCRA2? |
When cells are subjected toagents that damage DNA, Rad51foci assemble to activate the repairfunctions, with BCRA2 its involved in genomic stability, homolog of RecA |