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-11

• Native state is a dimer

• 8-stranded anitparallel beta barrel

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 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

Life Cycle of Epstein-Barr Virus3

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 EBV4

• Mononucleosis

• Polyclonal B Lymphoproliferative Disease (PLD)

• Burkitt’s Lymphoma

• Nasopharyngeal Carcinoma (NPC)

• Hodgkin’s Disease (HD)

Henrietta Lacks (HeLa)

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

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

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 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 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 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 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• 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

• 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.)

• 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

1 2 3

expected results

experimental 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

1= EBNA control DNA

2=(1)+ EBNA extract

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

Control + HeLa cells

5 4 3 2 1

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

Control + HeLa cells (#2)

• 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

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

Control + HeLa [DNA]

• 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

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

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)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

• 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

• 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

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= 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

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• 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

• Dr. Mascotti

• Dr. Lissemore

• Pierce

• Chemicon International