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

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

• Patrick Bruss
• Erin Shaneyfelt
• 2 May 2002

2
Crystal Structure of EBNA-11

• Native state is a dimer
• 8-stranded anitparallel beta barrel

3
EBNA-1 Bound to DNA1

• Blue central core of protein
• Goldadjacent alpha helicies which also contact
  DNA
• Binding site2

5-TAGCATATGCTA-3
3-ATCGTATACGAT-5
4
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

5
Life Cycle of Epstein-Barr Virus3
6
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.

7
Human Diseases Identified with EBV4

• Mononucleosis
• Polyclonal B Lymphoproliferative Disease (PLD)
• Burkitts Lymphoma
• Nasopharyngeal Carcinoma (NPC)
• Hodgkins Disease (HD)

8
Henrietta Lacks (HeLa)
Henrietta Lacks (HeLa) who died in 1951 from
cancer of the cervix5
9
Electrophoretic Mobility Shift Assay (EMSA)6

• apparent molecular weight of DNA-protein complex
  gt unbound DNA
• apparent mw of DNA-protein-antibody complex gt
  DNA-protein complex gt unbound DNA
• used to identify DNA binding proteins

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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.

11
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
  (lt150mM) 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

12
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

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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 centerslower 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
  (twohomodimers of each derivative
  oneheterodimer)

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Pros/Cons of EMSA7

• Advantages
• dont 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

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Procedure

• Followed protocol in Peirce EMSA handout2
• Binding Reaction
• components (total volume20uL)
• nuclease free water
• 10X Binding Buffer (Tris, KCL, DTT, pH 7.5)
• 50 Glycerol
• MgCl2
• Poly (dIdC) (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

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• 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.)

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• 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

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Control reaction
1 EBNA control DNA 2(1) EBNA extract 3 (1,2)
unlabelled EBNA control DNA
1 2 3
experimental results
expected results

• 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

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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
5 4 3 2 1

• 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

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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
1 2 3 4 5

• shift in HeLas 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

21
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
5 4 3 2 1

• 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

22
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

23
Electrophoretic Mobility Shift Assay (EMSA)6

• apparent molecular weight of DNA-protein complex
  gt unbound DNA
• apparent mw of DNA-protein-antibody complex gt
  DNA-protein complex gt unbound DNA
• used to identify DNA binding proteins

24
Nonspecific Binding and Antibody

• none of the bands are as intense as expected
  based on other labs
• other photons make extra spots that dont 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

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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)

• 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- didnt seem to work at all- low
  intensity indicates DNA loss

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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)
1 2 3 4 5 6 7 8 9 10

• 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

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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

28
References

• 1. Crystal Structure of EBNA-1. Department of
  Microbiology and Immunology, University of
  Rochester Medical Center. www.urmc.rochester.edu/s
  md/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_16
  0/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-filmmak
  er.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/Cecili
  aNorman.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.

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Acknowledgements

• Dr. Mascotti
• Dr. Lissemore
• Pierce
• Chemicon International