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Detection of Epstein-Barr Nuclear Antigen-1 in HeLa Cells Using Electrophoretic Mobility Shift Assay. Patrick Bruss Erin Shaneyfelt 2 May 2002. Crystal Structure of EBNA-1 1. Native state is a dimer 8-stranded anitparallel beta barrel. EBNA-1 Bound to DNA 1. Blue= central core of protein

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slide1
Detection of Epstein-Barr Nuclear Antigen-1 in HeLa Cells Using Electrophoretic Mobility Shift Assay

Patrick Bruss

Erin Shaneyfelt

2 May 2002

crystal structure of ebna 1 1
Crystal Structure of EBNA-11

Native state is a dimer

8-stranded anitparallel beta barrel

ebna 1 bound to dna 1
EBNA-1 Bound to DNA1
  • Blue= central core of protein
  • Gold=adjacent alpha helicies which also contact DNA
  • Binding site2=

5’-…TAGCATATGCTA…-3’

3’-…ATCGTATACGAT…-5’

binding in vivo 1
Binding in vivo1
  • two dimers shown
  • DNA sites separated by 3bp
    • same distance between EBNA-1 binding sites of oriP
  • proteins overlap and therefore conformational change in either protein, DNA or both
    • EBNA-1 is very rigid
    • DNA bends, thought to be crucial for DNA replication initiation
ebv associated proteins 3
EBV Associated Proteins3
  • after infection, 6 nuclear antigens are expressed
    • EBNA-1 maintains viral plasmid during latency and activates replication during the lytic cycle
    • EBNA-2 involved in immortalization of lymphocytes
    • EBNA-3(a-c) involved in transformation of human -lymphocytes
      • most information about mechanism is still unknown
  • EBNA-1 is the only proteins expressed in ALL EBV infected cells.
human diseases identified with ebv 4
Human Diseases Identified with EBV4
  • Mononucleosis
  • Polyclonal B Lymphoproliferative Disease (PLD)
  • Burkitt’s Lymphoma
  • Nasopharyngeal Carcinoma (NPC)
  • Hodgkin’s Disease (HD)
henrietta lacks hela
Henrietta Lacks (HeLa)

Henrietta Lacks (HeLa) who died in 1951 from cancer of the cervix5

electrophoretic mobility shift assay emsa 6
Electrophoretic Mobility Shift Assay (EMSA)6
  • apparent molecular weight of DNA-protein complex > unbound DNA
  • apparent mw of DNA-protein-antibody complex > DNA-protein complex > unbound DNA
  • used to identify DNA binding proteins
importance 7
Importance7
  • studies have shown that major control of genes and gene expression is done through DNA-protein interactions
    • e.g.: DNA replication, recombination and repair, transcription, RNA processing, viral assembly
  • to understand function of interactions, need to know information about structure of DNA-protein complexes, thermodynamics, and kinetics
  • Eletrophoresis mobility shift assay (EMSA) developed by Garner and Revzin to study these characteristics8.
conditions of binding 7
Conditions of Binding7
  • Protein-DNA complexes can be formed by mixing small amounts of protein with labeled DNA in low salt buffer
  • Formation of complexes can be influenced by many parameters
    • monovalent ion concentration-low ionic strength (<150mM) increase stability of interaction
    • presence of non-ionic detergents or carrier proteins-can stabilize product
    • time and temperature of binding reaction
    • protein concentration
    • type and concentration of competitor DNA
  • Nonspecific competitor such as poly(dA-dT) or poly(dI-dC) used to distinguish between specific and nonspecific binding
conditions of native polyacrylamide gel 7
Conditions of Native Polyacrylamide Gel7
  • mobility of complexes determined by size, charge, and confirmation of protein bound to DNA
  • composition of gel and electophoretic conditions can alter mobility and stability
    • higher conc. polyacrylamide stabilizes complex
applications of emsa analysis 7
Applications of EMSA Analysis7
  • Quantification
    • stoichiometric relationships between different complexes
  • Specificity of protein
    • DNA binding- perform experiment in presence of increasing amounts of unlabelled competitor
    • if competitor has high affinity binding site, will compete and decrease visible concentration of detectable complex
  • Equilibrium constants
    • obtained by mixing known amount of labeled DNA with increasing conc. of protein and construct standard binding curve
    • point at which 50% labeled DNA is bound with protein =Keq
  • Conformational changes of DNA
    • bent DNA molecules migrate slower than linear (also if bend is in center=slower than on end)
    • by creating DNA fragments which alter placement of DNA binding site, can study bending activity if protein
  • Stoichiometric analysis
    • number of protein that bind per DNA fragment
    • e.g.- using two different-sized derivatives of same protein-complexes will form three bands (two=homodimers of each derivative + one=heterodimer)
pros cons of emsa 7
Pros/Cons of EMSA7
  • Advantages
    • don’t need highly purified proteins
    • can resolve complexes that differ in protein and nucleic acid stoichiometry and/or conformation
    • easy to separate different species
  • Disadvantages
    • no information about sequence of binding site
    • difficult to adjust all parameters for complete optimization
procedure
Procedure
  • Followed protocol in Peirce EMSA handout2
  • Binding Reaction
    • components: (total volume=20uL)
      • nuclease free water
      • 10X Binding Buffer (Tris, KCL, DTT, pH 7.5)
      • 50% Glycerol
      • MgCl2
      • Poly (dI•dC) (in Tris, EDTA, pH 7.5)
      • 1% NP-40
      • DNA (biotinylated or not)
      • Protein/lysate
      • sometimes antibody9
    • incubate 20min at room temp
    • add loading buffer
slide16
6% Polyacrylamide gel
    • 0.5X TBE + 40% acrylamide + APS + TEMED
    • polymerize 1hr+
  • Load/Run gel
    • use 0.5X TBE buffer
    • ~200V, 20-25mA, about 20min.
  • Transfer to (+)nylon membrane
    • 0.5X TBE, ice cooled
    • 380mA
    • 30min.
  • UV crosslink (5min.)
slide17
Block/Wash
    • Lightshift Blocking Buffer
    • Lightshift Stabilized Streptavidin-Horseradish Peroxide Conjugate (filtered)
    • Lightshift 1X Wash Buffer
    • Lightshift Equilibration Buffer
  • Detection
    • Lighshift Luminol/Enhancer Solution
    • Lightshift Stable Peroxidase Solution
    • measure chemiluminescene by cooled CCD camera
      • 5-15min. exposure
control reaction
Control reaction

1= EBNA control DNA

2=(1)+ EBNA extract

3= (1,2)+ unlabelled EBNA control DNA

  • Loading dye was omitted from lanes 1 and 2 and therefore they did not have enough glycerol and the DNA diffused away
  • Still see expected shift in lane 2 due to EBNA DNA-protein complex

1 2 3

experimental results

expected results

control hela cells
Control + HeLa cells

1= EBNA control DNA

2=(1)+ EBNA extract

3= (1,2)+ unlabelled EBNA control DNA

4=(1)+ Active Motif HeLa

5=(1)+ Dr. Mascotti HeLa

  • gel did not run correctly due to buffer dilution error
    • stopped immediately after lanes entered gel
  • no shift for EBNA control (lane2)
  • binding to site for both HeLa samples
  • binding of approximately same size protein

5 4 3 2 1

control hela cells 2
Control + HeLa cells (#2)

1= EBNA control DNA

2=(1)+ EBNA extract

3= (1,2)+ unlabelled EBNA control DNA

4=(1)+ Active Motif HeLa

5=(1)+ Dr. Mascotti HeLa

  • shift in HeLa’s about same size as EBNA shift
    • could indicate presence of EBNA
    • or another protein of similar size that recognizes binding site
  • upper bands= nonspecific binding

1 2 3 4 5

control hela dna
Control + HeLa [DNA]

1= EBNA control DNA

2=(1)+ EBNA extract

3= (1,2)+ unlabelled EBNA control DNA

4=(1)+ Active Motif HeLa

5=(1)+ Dr. Mascotti HeLa

  • EBNA control shift is missing
    • could be due to less template available to bind
    • forgot to put in reaction
  • Bands in HeLa lanes are the ones that match EBNA shift
    • most intense bands from previous gel
    • other bands are gone due to lower concentration of protein

5 4 3 2 1

preliminary conclusions
Preliminary conclusions
  • EMSA works correctly and detects DNA to at least 1femtomole
  • Found a few proteins in HeLa cells that recognize EBNA binding site
  • One of these proteins in each HeLa sample matches shift of EBNA protein-DNA complex
  • Could be specific or unspecific binding
  • Could be EBNA or a different protein that happens to have a similar size
electrophoretic mobility shift assay emsa 623
Electrophoretic Mobility Shift Assay (EMSA)6
  • apparent molecular weight of DNA-protein complex > unbound DNA
  • apparent mw of DNA-protein-antibody complex > DNA-protein complex > unbound DNA
  • used to identify DNA binding proteins
nonspecific binding and antibody
Nonspecific Binding and Antibody
  • none of the bands are as intense as expected based on other labs
  • other photons make extra spots that don’t have DNA
    • pockets of substrate between the membrane and saran wrap
    • contamination that could have gotten some DNA and substrate bound
  • not sure about the shifts or lanes present to get reliable results
possible interpretation 1

1= biotin-control DNA

2= (1) + extract EBNA

3= (1,2) + unlabeled DNA

4= (1) + Active Motif

5= (1,4) + unlabeled DNA

6= (1,4) + antibody

7= (1) + Dr. M. HeLa

8= (1,7) + unlabeled DNA

9= (1,7) + antibody

10= (1,2) + antibody (control)

Possible Interpretation #1
  • lane 3- contamination ? (should not have a shift)
  • bottom of dye front ran off gel
  • intensities of HeLa inverted from other trials
  • lane 5- could be a bit of chasing- would indicate specific binding (but not the band that matches EBNA shift)
  • lane 6- could be a little higher, but is smeared
  • lanes 9,10- didn’t seem to work at all- low intensity indicates DNA loss
possible interpretation 2

1= biotin-control DNA

2= (1) + extract EBNA

3= (1,2) + unlabeled DNA

4= (1) + Active Motif

5= (1,4) + unlabeled DNA

6= (1,4) + antibody

7= (1) + Dr. M. HeLa

8= (1,7) + unlabeled DNA

9= (1,7) + antibody

10= (1,2) + antibody (control)

Possible Interpretation #2
  • only see one shift from extracts
  • unlabeled DNA control still did not work
  • lane 10- could be supershift from antibody
  • lane 8,9- still did not work

1 2 3 4 5 6 7 8 9 10

conclusions
Conclusions
  • None of the earlier conclusions were disputed
    • there is binding in the HeLa lysates that match shift with EBNA
    • could be specific or nonspecific
  • Antibody could be binding and there is no change in shift due to charge interactions, or conformational changes that counteract the additional weight
  • Need to run the last experiment again to get reliable results
references
References
  • 1. “Crystal Structure of EBNA-1.” Department of Microbiology and Immunology, University of Rochester Medical Center. www.urmc.rochester.edu/smd/mbi/grad2/herp99BB6.html. 2002. (25 April 2002).
  • 2. “Lightshift Chemiluminescent EMSA Kit.” Pierce. Rockford, IL, 2002.
  • 3. Solomon, Julie, Carla Fowler, and G. Cooper. “Epstein-Barr Virus.” www.brown.edu/Courses/Bio_160/Projects2000/Herpes/EBV/Epstein-Barr.html. Brown University, 2002. (25 April 2002).
  • 4. Kang, Myung-Soo, Ciu Chun Hang and Elliot Kieff. “Epstein-Barr virus nuclear antigen 1 activiates transcription from episomal but not integrated DNA and does not alter lymphocyte growth.” Proceedings of the National Academy of Sciences, USA. 98(6), 15233-15238, 2001.
  • 5. Potier, Beth. Harvard University Gazette. www.news.harvard.edu/gazette/2001/07.19/04-filmmaker.html. President and Fellows of Harvard College, 2002. (25 April 2002).
  • 6. Lissemore, J. “EMSA.” Molecular Genetics (BL465), John Carroll University, 24 April, 2002.
  • 7. Norman, Cecilia. “Electrophoresis mobility shift assay (EMSA).” SLU, Uppsala. www.plantae.lu.se/fskolan/arabidopsistexter/CeciliaNorman.html, (30 April 2002).
  • 8. Garner, M M. Rezvin, A. (1981) Nucl. Acids Res., 9 (13), 3047-3060
  • 9. “Mouse Anti-Epstein Barr Virus Nuclear Antigen (EBNA-1) Monoclonal Antibody.” Chemicon International. CA, 2002.
acknowledgements
Acknowledgements
  • Dr. Mascotti
  • Dr. Lissemore
  • Pierce
  • Chemicon International