Mbb 331 Lec 9-10

Front 1

What's a transition mutation

Back 1

Purine to purine mutation

Front 2

What's a transversion mutation

Back 2

pyrimidine to purine or vise versa

Front 3

What's EMS damage

Back 3

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

Front 4

Name two types of direct repairs and explain them

Back 4

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.

Front 5

What type of repair is DNA glycosylase? Describe it.

Back 5

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.

Front 6

Describe AP endonuclease

Back 6

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

Front 7

What are the two steps to Nucleotide excision repair

Back 7

Incision - endonuclease cleaves both sides of damaged base (UvrABC excinuclease)

Synthesis- replace with new base by polymerase/ligase seals nick

Front 8

Explain NER in E.coli

Back 8

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

Front 9

Explain NER in humans

Back 9

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

Front 10

Describe Mismatch repair in E.coli (Mut family of enzymes)

Back 10

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

Front 11

Describe how double-stranded break repair happens (NHEIJ non-homologous end joining)

Back 11

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

Front 12

3 classes of Genetic recombination

Back 12

1. generalized/homologous recombination

2. Site-specific recombination

3. Transposition of mobile elements

Front 13

Describe the RecBCD pathway in E.coli

Back 13

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

Front 14

What are homologous in eukaryotes and E.coli in terms of the RecBCD pathway? What induces double stranded break during meiosis?

Back 14

Homologs of Rec (RecBCD, RecA, RuvA-B-Chomologs) and Ruv proteins

Spo11, MRX complex - induces double stranded breaks during meiosis

Front 15

What is RecA's participation presynapsis? How/what protein aids in this?

Back 15

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

Front 16

What is RecA's participation in synapsis?

Back 16

Alignment of complementary sequencesin ss and ds DNAs , alignment unlikely to be Watson-crick pairing due to low melting temp

Front 17

What is RecA's participation postsynapsis?

What experiment showed this?

Back 17

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

Front 18

Two types of DNA damage

Back 18

structural distortions or single base pair

Front 19

name single base pair change examples

Back 19

2 types of deanimation: cystine to uracil and adenine to hypoxanthine (created by guanine deaminase)

and base pair change caused by errors in replication

Front 20

Do single base pair damage disrupt transcription? Which ones do?

Back 20

no, but its damaging to future generations, structural do disrupt transcription and replication

Front 21

How are pyrimidine dimers formed?

Back 21

Through structural base pair damage, by UV radiation, causing covalent linking to occur

Front 22

Name 3 types of structural damages

Back 22

pyrimidine dimerization by UV, depurination (missing bases), and base alkylation (addition of bulky groups to products)

Front 23

Electron rich centres in DNA are hot spots for what type of structural damage? why?

Back 23

alkylation, because the electrophile (wants electrons) so it gets it by attacking the phosphodiester bond

Front 24

Who won the Nobel Prize for DNA repair mechanisms?

Back 24

Tomas Lindahl, Paul Modrich, Aziz Sancar

Front 25

What is Direct Repair?

Back 25

damaged base is not removed, it is repaired on site

Front 26

Describe the Human BER pathway

Back 26

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

Front 27

What is XP? and what is it caused by?

Back 27

Xerodermapigmentosum: UVexposure leads to skincancer

XP is caused byexcision repair defects– XP human genes aresimilar to yeast RADexcision repair pathway

Front 28

How does XP disorder occur?

Back 28

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

Front 29

how does the repair system“know” which base to remove on which strand?

Back 29

THE MISMATCH REPAIR SYSTEM IDENTIFIESORIGINAL METHYLATED STRAND (because the newlysynthesized strand is not yet methylated)

Front 30

what is a retrieval system? what protein does it require?

Back 30

they carry out post-replication/recombination repair, Gap is filled by “retrieving” the correspondingsequence from the normal duplex viahomologous recombination,

requires recA

Front 31

what is the SOS response? how is it activated in E.coli?

Back 31

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

Front 32

2 ways double strand breakage can be repaired?

Back 32

1. Similar to bacterial retrieval repair involving homologousrecombination (S & G2)

2. Non-homologous end joining (NJEJ) in G1

Front 33

what binds to a NITROCELLULOSE FILTER BINDING ASSAY?

Back 33

ssDNA binds to the nitrocellulose filter, dsDNA does not,but if dsDNA is bound to the protein, it binds

Front 34

How does a GEL MOBILITY ASSAY detect an interaction between a protein and DNA?

Back 34

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)

Front 35

What is the process of DMS footprinting?

Back 35

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.

Front 36

What is CHROMATIN IMMUNOPRECIPITATION(ChIP)? How does it work?

Back 36

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.

Front 37

What is YEAST TWO-HYBRID SYSTEM? How does it work?

Back 37

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.

Front 38

How can you tell the difference between splice (crossover) products and patch (noncrossover) products? (homologous recomb)

Back 38

spliced is if the resolution of the holiday junction are not the same color, its patched if they are

Front 39

Does RecBCD nick at chi sites to initiate recombination? what was the experiment?

Back 39

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

Front 40

What is a recombination hot spot? Can they function in both directions?

Back 40

Hotspot is Chi sequences that promote recombination/crossover

No, Chi sequences are non-symmetric (which is why theyfunction only in one orientation):

Front 41

Chi sites have an effect on what directly?

Back 41

recombination frequency

Front 42

What is RecBCD's role?

Back 42

RecBCD helicase/nucleaseprocesses broken DNA moleculesto generate regions of ssDNArequired by RecA to initiate strandexchange

Front 43

What's the difference between RecB and RecD?

What is the result of RecB's movement?

Back 43

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.

Front 44

RecC does what?

Back 44

Recognizes Chi sites, Cleaves specific DNA strands at the holliday junction to finish recomb.

Front 45

Where does RuvA bind? RuvB? What does RuvB do? Experiment that proved this?

Back 45

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

Front 46

In eukaryotes, programmed generation of DS DNA breaks occurs when and mediated by?

Back 46

programmed generation of DSB during meiosisis mediated by Spo11 and the MRX complex

Front 47

In eukaryotes, what is the homologous recomb pathway?

Back 47

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

Front 48

Mutations in what are thought to be responsible for half familial breast cancers?

Back 48

BRCA2

Front 49

What does Rad51 do? What is it a homolog of? What happens when it interacts with BCRA2?

Back 49

When cells are subjected toagents that damage DNA, Rad51foci assemble to activate the repairfunctions, with BCRA2 its involved in genomic stability, homolog of RecA