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BMSC320 > DNA Binding > Flashcards

DNA Binding Flashcards

1
Q

Small ligands (drugs) can bind DNA by:

A

Intercalation, groove binding, covalent interaction

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2
Q

What is intercalation?

A

Small ligands bind DNA in between base pairs

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3
Q

What characteristics do intercalating agents need to have?

A

cyclic, hydrophobic, aromatic, planar, small (CHAPS)

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4
Q

why is ethidium bromide (EtBr) a good intercalating agent? (5 reasons)

A
  • Used as a fluorescent dye for DNA
  • has high affinity for DNA because EtBr is ⊕ and DNA phosphate backbone is ⊖
  • transient relaxation of base stacking allows its entry between bases
  • intercalation is stabilized by stacking interactions between it and nucleotides
  • is weakly fluorescent on its own due to water, but fluorescence increases 20x in DNA
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5
Q

Effects of EtBr as an intercalating agent

A
  • unwinds B DNA from 36° turn to 10° turn
  • a saturated molecule is ~ 27º longer due to unwinding and increased rise between bases
  • at 1mM solution EtBr can occupy 57% of sites, so side by side intercalation is possible but more likely every second site is occupied by EtBr
  • binding distortion can affect unbound regions by causing extra supercoiling
  • stretched DNA is more likely to have frameshift mutations during replication
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6
Q

Main lab use for EtBr as intercalating agent

A

visualize DNA or RNA in agarose gel

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7
Q

Secondary lab use for EtBr

A

separation of plasmid DNA from genomic DNA (which is linear and too long not to break)
- use density gradient ultracentrifugation
- genomic (linear) DNA binds more EtBr and density decreases
- Plasmid (circular) DNA binds less EtBr due to excessive supercoiling and is more dense
- plasmid DNA sinks deeper than genomic DNA
- celsium density gradient forms at very high speeds (1/2 million x gravity)

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8
Q

Intercalation drug mechanism

A

Intercalation induces structural DNA changes that can inhibit replication, transcription, and topoisomerase binding

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9
Q

EtBr for african sleeping sickness

A

anti-trypanosomal drug used in cattle at 1mg/kg
- affects parasites because they have more circular DNA than cows/humans
- impairs mitochondrial DNA replication in parasite
- can theoretically drink thousands of liters of gel staining strength and have a non-toxic dose

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10
Q

Intercalating drugs: Anthracyclines

A
  • derived from natural antibiotics produced by Streptomyces
  • planar molecule that intercalates between alternating purine/pyrimidines
  • has selective toxicity against rapidly-proliferating cells
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11
Q

Daunorubicin timeline

A

Daunorubicin was first anthracycline drug for cancer
- in the 50’s it’s discovery as antibiotic showed action against mouse tumors
- in the 70’s it was used against leukemia and breast cancer

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12
Q

Daunorubicin mechanism

A
  • prevents replication and transcription by interfering with topoisomerase II re-ligation in free-radical production
  • can intercalate 1 in 3 bases
  • causes 8° - 11° unwinding with preference for AGC, TGC, ACG, or TCG sequences
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13
Q

Daunorubicin side effects

A

hair loss, bone marrow suppression, nausea, reversible (dose dependent) heart damage at greater than 600mg/m²
typical dose 45mg/m²

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14
Q

What is groove binding?

A

Compounds may bind major or minor groove of DNA depending on the size of each groove

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15
Q

characteristics of groove binding ligands

A

crescent shaped or flexible to accommodate the groove in the target DNA

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16
Q

How do small ligands associate with DNA during groove binding?

A

associations typically displace the hydrogen shell
may be through ionic bonds, hydrogen bonds, and van der Waals

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17
Q

5 common minor groove binders

A
  • spermine: stabilizes and compacts DNA
  • neotripsin: abx
  • distamycin A: anti-cancer
  • hoechst 33258: fluorescent DNA stain
  • DAPI: fluorescent DNA stain
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18
Q

What is neotripsin and how does it work?

A
  • Antibiotic that shows specificity to AT rich tetramiric sequences on crystallography
  • does not show binding to ssDNA or dsDNA because the shape is different
  • high affinity for DNA prevents binding of helicases and isomerases
  • potential to be developed for anti-viral/anti-cancer
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19
Q

What are Hoechst stains and how do they work?

A
  • Hoechst 33258 and 33342 are UV activated blue-fluorescent dyes used as nuclear stains
  • Fluorescence is enhanced by proper DNA binding
  • AT rich sequences give double the fluorescence as GC rich sequences
  • can use to stain live cells to see the nucleus
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20
Q

Hoechst stem cell example

A
  • Hoechst 33342 is actively pumped out of hematopoietic stem cells faster than other blood cells and therefore might have different nuclear/cytoplasmic ratio
  • Flow cytometry can measure DNA bound dye (blue) and unbound dye (red) to pick out stem cells
    extracting the “side population” where the stem cells are isolated
  • can repopulate bone marrow and immune system in mice, primates, and humans
    some cancer cells can be quantified using HeLa cells
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21
Q

Timeline of covalent DNA binding compound mustard gas

A

late 1800s: organic chemists produce sulfur mustard (impurities give it a mustardy/garlicy smell)
1913: organic chemist in Berlin comes up with a better way to make it but lab accident causes severe burns, German government weaponizes it as mustard gas
1917/18: used in WWI by germans and then British
1919: autopsies on mustard gas fatalities show low white blood cell count (= impaired hematopoiesis)
1942+: mustard gas derivatives (mustargen) being studied as chemotherapeutic agents

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22
Q

Mustargen side effects

A

-severe blistering and burns
-temporary blindness
-if inhaled, blistering + fluid in lungs (drown from the inside)
- if swallowed, nausea, vomiting, diarrhea
- death

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23
Q

Mustargen chemical reaction

A

In aqueous solution, loses one Cl to produce cyclic etheneimmonium ion
- is highly reactive and has specificity for N7 of guanine but can react other places as well
-alkylated guanine (attached to mustard gas) can have:
1. depurination leading to DNA breakage
2. cross strand crosslinking: G-alkylating agent/mustard gas-G
3. mispairing with T

24
Q

What are the 2 possible results of mustargen crosslinking with two guanines?

A

If the crosslink occurs between two Gs on the same strand, they can no longer properly base pair because they’re locked together.
If crosslink occurs between the two strands, DNA can no longer be unwound

25
Q

Chemotherapy with alkylating agents

A

Cancer cells divide quickly so don’t have time to repair mutations
alkylating agents produce mutations:
-if fixed, no problem from the cell because normal cells will detect mutation and fix it
-if unfixed, may render cell non-viable or cause apoptosis (many cancer cells have this pathway turned off)

26
Q

Assumption for intelligent drug design

A

Assumed that better differential toxicity could come from adding cyclic phophamide group to mustargen which should produce non-reactive prodrug that is unable to alkylate until the N-P bond is cleaved

27
Q

What did testing of pro-drug find

A

-drug is activated in liver to aldophosphamide
-cells with high levels of aldehyde dehydrogenase (bone marrow and intestinal epithelium) break down the active form and are safe
-cancers with low ALDH are alkylated and die
-also targets tissues with autoimmune inflammation
-less toxic than mustargen, can give 100x more without dying

28
Q

Compare DNA/Drug interactions by intercalation, groove binding, and covalent binding

A
  • intercalation binds one or more locations by slipping in between bases and being stabilized by stacking interactions. decreases twist and increases rise. ex. is EtBr for fluorescence, daunorubicin for cancer
  • groove binding agents are curved and flexible and lie across the groove. more minor discovered than major. some stabilize DNA, some are antibacterial/anticancer, some are non-toxic and fluorescent. ex is neotripsin antibiotic, hoescht stain for fluorescence
  • covalent binding contains reactive centre that modifies DNA with some specificity and can lead to problems with DNA replication fidelity
29
Q

Difference between specific and nonspecific DNA/protein interactions

A

nonspecific: protein binds DNA such as histones with basic amino acids bind to phosphate groups by charge interaction
specific: protein binds to a DNA sequence or structue

30
Q

Steps for hypothetical scenario where we have evidence that the product of gene x controls expression of gene y

A

1: discover the region of the gene that protein x binds
2: show specificity for that gene region
3: use that region to determine the exact sequence bound
4: determine structure of DNA-protein complex

31
Q

how does chromatin immunoprecipitation (CHiP) work?

A
  • proteins bound to DNA are often lost during DNA isolation, so crosslinking reaction is done to fuse proteins to DNA so they stay stuck to the specific sequence
  • antibodies are used to precipitate out the proteins and the attached DNA sequence
  • since crosslinking is reversible it only isolates the DNA sequence of interest
  • only provides a long sequence of DNA
32
Q

how does electrophoretic mobility shift assay (EMSA) work?

A

-trims down region of DNA sequence necessary to bind protein of interest
-run DNA through gel, DNA is labeled and now called probe
-probe is added to protein of interest which makes the molecule heavier and move more slowly (shift)
-use a competitor to see if protein of interest will bind more tightly to competitor or probe
- if competitor works well, less protein binds probe = more probe at the bottom of gel
- if competitor works poorly, more protein binds probe = less probe at the bottom of gel
- mutate competitor by changing base pairs to see if protein will bind to it or probe better
- use antibody specific to protein of interest to see if it is bound to the probe
- if antibody binds the probe-protein-antibody complex migrates slower = supershift
- if antibody doesn’t bind the probe-protein will migrate faster

33
Q

How does DNAse I Footprinting work?

A

-Label DNA sequence of interest on only one strand and have one set of naked DNA and one set of DNA bound to protein of interest
-slightly digest DNA by adding enough DNAse to get about 1 cut per molecule
-Run DNA in denaturing gel that makes it single stranded and we can only see the strand thats labeled
-naked DNA should have a cut at every position so it looks like a ladder
-DNA bound to protein will produce a footprint (gap in ladder due to protein blocking enzyme from cutting DNA)
-sequence may be more protected on one strand than the other or sequence may shift some bases left or right

34
Q

How does x-ray crystallography work?

A

use diffraction patterns to get electron density using Fourier Transform to find exactly what amino acids of the protein are bound to the DNA sequence

35
Q

3 main ways that proteins bind DNA

A
  1. basic amino acids have ⊕ charge, attach to ⊖ charge phosphate backbone
  2. some amino acid side chains fit into a groove and make specific recognition pairings with the side of a base pair
  3. some flexibility in the protein and in DNA allow for bending and folding to make more stable contacts
36
Q

Which groove will have less discrimination during protein binding?

A

Minor groove because it does not have enough specificity

37
Q

Which pair is more distinguishable for groove binding?

A

G-C pairs are more distinguishable than A-T pairs

38
Q

How many DNA binding proteins are there?

A

close to 3000, most fall into 4 superclasses

39
Q

80% of all DNA-binding domains fall into which 4 major types?

A

Helix-turn-helix, Zinc fingers, leucine zipper, and helix-loop-helix

40
Q

what is the structure of the helix turn helix (HTH) DNA binding domain?

A

alpha helix, then flexible linker, then another alpha helix for about 20 amino acids in total

41
Q

where is the HTH domain?

A

first alpha helix fits into major groove with sequence specificity (recognition helix)
remaining protein (2nd helix) outside this domain adds to specificity and stability

42
Q

HTH Tryptophan repressor example

A

turns off synthesis genes for trp when trp is present

homodimer bind operator through dual HTH domains and turns off the trp operon in E. coli when bound to trp

when trp is present, there is proper distance in binding sites which allows DNA to bind and turn off trp operon
when trp is absent it is less bound to protein which changes distance and destabilizes binding to DNA which allows operon to be turned on

43
Q

HTH domain homeobox gene example

A

homeobox genes act as transcription factors and bind by HTH domains. they are master control genes for anterior to posterior pattern regulation in animals
(first one turned on in anterior part of body near head, last one gets turned on in posterior part of body near tail)
controlled by having chromatin turn everything off and slowly reveal genes as it gets to posterior part of body.
if posterior gene is transcribed more anterior then the structures will develop at the wrong spots.
in segmented animals if genes are expressed too early or too late, can have legs where antennae should be

44
Q

What is the structure of zinc finger DNA binding domain?

A

usually one protein that has multiple domains that bind
- about 20-30 amino acids in length
- defined positions of particular amino acids will hold a zinc atom
-most common is the C2H2 zinc finger: zinc ion is held by 2 cysteines and 2 histidines

45
Q

Where do the zinc finger domains bind?

A

They have specific binding along the major groove
-specificity can’t be determined from amino acid sequence, must be done experimentally
-alpha helix-beta strand structure can lie across the major groove to bind DNA
-common to have multiple zinc fingers in succession in one protein to provide binding affinity and stability

46
Q

Structure of TFIIA of Xenopus (frog)

A

has 9 zinc fingers
1,2,3 and 7,8,9 wrap around major groove and 4,5,6 are free.
there are 10bp intervals between sets of fingers
each triple finger binds a 14 base pair sequence

47
Q

What is the structure of leucine zipper DNA binding domain?

A

Dimer of elongated alpha helices that can be homo or hetero dimer
- one monomer will run along the front face of DNA and the other along the back
- N-terminus in enriched for basic amino acids and will contact DNA
- C-terminus contains repeating leucines of every 7th amino acid
-standard alpha helix is 3.6 aa’s per turn, but leucine zipper is overwound to 3.5 aa’s per turn
- every 7th amino acid = 2 turns of the alpha helix

48
Q

How does the leucine zipper bind to DNA?

A

pairs of hydrophobic leucines pair up to stabilize coiled-coil of two alpha helices wrapped together
- initially thought that leucines were staggered (like a zipper) but they actually interact directly like buttons
-a typical recognition site is 4bp per contact
-sequence specificity comes from the part of the dimer that touches DNA, not the zipper part
- if it is a heterodimer, each monomer may have a different specificity sequence

49
Q

What is the structure of the helix-loop-helix (HLH) binding domain?

A

a pair of proteins that have specificity through two alpha helices
-instead of being held by paired leucines, the second helix is amphipathic with the dimerization through the hydrophobic faces of the helix
-can act as homodimers or heterodimers

50
Q

Combinatorial gene expression

A

because most motifs are a dimer bound to DNA sequences, homo and heterodimers give slightly different specificities and more control over gene expression
-from 3 proteins that can form dimers we can get six novel binding sites recognized
-results in diverse gene expression

51
Q

compare HTH, zinc finger, leucine zipper, and HLH binding domains

A

HTH: dimer with a recognition/specificity helix and a stability helix
zinc fingers: multiple domains possible per protein, each bind 4/5 bp in the major groove
leucine zipper: coiled coil of 2 entwined alpha helices (dimers) paired at leucines that bind the front and back faces of the major groove
HLH: interrupted coiled coil held together at hydrophobic faces (dimers)

52
Q

What is important about binding by shape specificity?

A

There is less understood about proteins exhibiting strong DNA binding affinity but poor sequence specificity, and it’s harder to study because sequence and shape can be linked

53
Q

Shape specificity example of insulator protein su(Hw)

A

su(Hw) is a protein that insulates gene activation and restricts it to only a single loop of DNA. It binds specific sequence in the central box, but stability depends on the presence of polyA:T runs to either side. A mutation to 2 GC pairs to the center of each run destabilizes protein:DNA interaction because it removes the ability of the DNA to bend around the protein

54
Q

Shape specificity example of BRCA1 (familial breast cancer gene)

A

Stabilizes ds breaks and is important for genomic stability. If mutated, may be cancer causing. MCRA -/- mouse cells show increased chromosome breakage and susceptibility to mutagens. Since there is no sequence to breaks (is random and may not affect a certain sequence), BRCA1 should bind broken chromosomes based on shape/structure, not sequence

55
Q

EMSA results on BRCA1

A

If it has structural specificity it should respond to EMSA competition experiments.
Results conclude specificity is cruciform> C:C>bulge> G:T (linear)

BMSC320 (9 decks)

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