12.18.09 Theis Lab Group Presentation


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My end of the semester Theis Lab group presentation

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  • -Many repair mechanisms work to identify damage of transcribed genes and therefore, genes are much more likely to be repaired after they have been transcribed -Persistence of unrepaired DNA damage can cause mutations and other detrimental factors that can lead to cancer, cell death, aging, and Cockayne’s syndrome -Symptoms of Cockayne’s syndrome impaired nervous system development and premature aging -I am studying the DNA repair protein, Mfd
  • -Mfd is made up of MfdN and MfdC which are tethered to each other by a linker between them -The are MfdN, which is composed of domains 1 through 3 and MfdC which is composed of domains 4 through 7. MfdC and MfdN are linked together by the linker at domain 3 and domain 4 -I am studying the role of MfdC in transcription coupled DNA repair -MfdC’s role is to bind to DNA during transcription, and to act as an ATP motor for a conformational change
  • -To understand how Mfd works, I have to give a bit of background on another enzyme, RNA polymerase -When RNA polymerase attempts to transcribe damaged DNA, it will sometimes get stalled and just sit on the DNA -Here is where Mfd comes in -Mfd then recognizes the stalled RNA polymerase, then, the MfdC subunit binds to the DNA behind (upstream of) RNA polymerase. -MfdC uses its ATP motor to cause MfdC and MfdN to open up -The theory is that then MfdN moves to sort of push RNA polymerase off of the DNA -Then, Mfd somehow calls in DNA repair enzymes UvrA, UvrB, nuclease and ligase to repair the damaged DNA
  • -My goal is to obtain the structure of MfdC when ATP is bound to it and when DNA is bound to it and hopefully gain some insight in the issues above.
  • -We use a mutant called ATHook-MfdC, which contains a residue that binds well with AT rich areas of DNA -Mike Murphy and Peng Gong found that, compared to Full-Length Mfd, ATHook is superior in terms of activity, binding, and stimulation by DNA
  • -This is my final purification procedure that we have developed over multiple grow-ups -It’s proven difficult to get MfdC to a pure enough state and at a high enough concentration where we can find consistent crystal results and therefore we need to go through many rigorous steps to get there. -MfdC at high concentrations when expressing in cells can be toxic to the cell, making it a particularly hard protein to express in high amounts.
  • We did another grow up, this time in 4L to try to get repeatable results in the crystal tray Several samples are missing from the gel  we forgot to take some samples along the way The lysate sample precipitated in the loading dye. It was sitting in the dye for a long time and had been cooked twice You can see most of our protein came off in NiE2, that was a 4.5mL fraction Elution buffer is .5M NaCl, 10mM Tris pH 8.2, 250mM Imidazole We then desalted (not shown) and diluted the fractions to 250mM NaCl to load onto the Q column MfdC does not bind to the anion exchange resin so it comes out in the FT FT fractions are from 10-12mL We then eluted contaminant proteins in 4, 2mL fractions designated QE1-QE4 SDS Samples are 7uL
  • This was a 1mL heparin column It is not very pure, most protein came out in Hep E2 and E3 Each of these are 1mL fractions Hep E2 was taken and loaded onto the sizing column it was at ~2mg/ml SDS Samples are 7uL
  • Hep E2 was taken from the previous gel and loaded onto the sizing column There was a bubble in the UV detector Each fraction is 3.5mL It came out relatively pure SDS Samples are 7uL
  • We collected fractions 29-39 from the sizing column, brought them down to 250mM salt and reapplied them to a 1mL heparin column This concentrated our protein (relatively speaking of course) E2 was about 1mg/ml from the Garman lab spec SDS samples are 7uL
  • Off of our final heparin column we took Hep E2 and buffer exchanged it into .5M Ammonium Acetate; a volatile salt. We then added MgCl2 for a target concentration in the well of 4mM, and AMPpnp for a target concentration in the well of 2mM We used a 1:6 ratio of precipitant to protein The volatile buffer caused the drop to concentrate to 1microliter We got a crystal in C4, which contained .2M Sodium Acetate Trihydrate, .1M Sodium Cacodylate pH 6.5, and 3% w/v PEG 8000 The crystal is about 20micrometers
  • NADH absorbs light at 340nm Pyruvate Kinase and Lactose dehydrogenase are fast enzymes so we can accurately measure the rate of ATP hydrolysis by measuring the rate of disappearance of NADH
  • In the past, I have had problems with very low activities, over the summer I figured out that the Ph of our buffers were all off by like 2 ph units. The ATPase assay was done on the DU-800 Spectrophotometer in the Heuck lab at 37 degrees C The activity was measured for 10 minutes, but the assays get wiped out quickly We ignored the nonlinear activity where the substrate concentrations were very low This graph shows how ATHook-MfdC is stimulated twice as much by DNA Concentration of stock protein was about .150mg/mL
  • 12.18.09 Theis Lab Group Presentation

    1. 1. <ul><ul><li>Chris Sandifer </li></ul></ul>Crystallization and ATPase Activity of MfdC Theis Lab
    2. 2. DNA damage, cancer, aging DNA Damage DNA Repair Mutations Replication Errors Persistent DNA Damage Genomic Instability Cancer Cell death Aging Replication Transcription and DNA repair <ul><li>Transcribed genes are preferentially repaired </li></ul><ul><li>Defect in transcription-coupled repair in humans leads to Cockayne’s syndrome </li></ul>Images courtesy of Karsten Theis
    3. 3. Sequence and structure of Mfd Darst and coworkers (2006) PDB ID 2EYQ Images courtesy of Karsten Theis
    4. 4. Mfd, the bacterial TRCF RNA polymerase stalled at DNA damage is recognized by Mfd pre-incision complex (UvrB “padlock” bound to damaged DNA) RNA, RNAP Mfd UvrA 2 UvrA 2 UvrB RNA Pol Mfd COUPLING <ul><li>Removal of RNA polymerase </li></ul><ul><ul><li>Make DNA damage accessible </li></ul></ul><ul><ul><li>Rescue arrested transcription (transcription regulation) </li></ul></ul><ul><li>Recruitment of DNA repair enzymes </li></ul><ul><ul><li>Has to be faster than next polymerase arriving at site </li></ul></ul>Images courtesy of Karsten Theis UvrB C N
    5. 5. Goal: Obtain crystal structures of MfdC:ATP and MfdC:ATP + DNA <ul><li>Optimal DNA length for crystallization </li></ul><ul><li>Identify DNA-binding residues </li></ul><ul><li>Conformational changes </li></ul>7 5 4 6 DNA groove Proposed path of DNA
    6. 6. My MfdC Mutant <ul><li>Low ATPase activity (turnover ~10/min) </li></ul><ul><li>No robust DNA binding </li></ul><ul><ul><li>Need to add non-hydrolysable ATP analogs </li></ul></ul><ul><ul><li>Need to use DNA of >100 bp </li></ul></ul><ul><li>ATPase activity hardly stimulated by DNA (1.2-fold) </li></ul><ul><li>No ATP binding site available in apo conformation </li></ul>Full Length Mfd: ATHook-MfdC: <ul><li>High ATPase activity (observed turnover ~100 to 150 mM ATP/min/mM Enzyme) </li></ul><ul><li>ATHook binds DNA strongly </li></ul><ul><ul><li>Binds to AT rich areas of DNA very well </li></ul></ul><ul><ul><li>Can use DNA of < 100 bp </li></ul></ul><ul><li>ATPase activity substantially stimulated by DNA (~2.0-fold) </li></ul>
    7. 7. Grow Up & Purification of MfdC <ul><li>T7 I q Cells with ATHook-MfdC </li></ul><ul><li>Incubate at 37°C, Induced at 30°C </li></ul><ul><li>Ni Affinity Column </li></ul><ul><li>Desalt </li></ul><ul><li>Anion Exchange Column </li></ul><ul><li>Heparin Affinity Column </li></ul><ul><li>Sizing Column </li></ul><ul><li>1mL Heparin Affinity Column (To concentrate) </li></ul>
    8. 8. SDS-PAGE Nickel and Anion Exchange 4L Grow Up NiFT NiW NiE1 QE1 QFT4 NiE4 NiE3 NiE2 Mark QFT2 QE3 QE4 QE2 80
    9. 9. First Heparin Column 80
    10. 10. Sizing Column 80 38
    11. 11. Final Heparin Column Final Heparin Column Mark 80 HepFT HepW HepE7 HepE6 HepE5 HepE4 HepE3 HepE2 HepE1
    12. 12. Crystallization of MfdC with MgCl 2 and AMPpnp <ul><li>Concentrated in drop with volatile buffer, Ammonium Acetate .5M </li></ul><ul><li>1:6 Ratio of Precipitant to protein </li></ul><ul><li>Crystal formed in .2M Sodium Acetate Trihydrate, .1M Sodium Cacodylate pH 6.5, 3% w/v PEG 8000 </li></ul>~20 μ M
    13. 13. ATPase Assay MfdC ATP ADP+P i Pyruvate Kinase Phosphoenolpyruvate Pyruvate Lactate Dehydrogenase Lactate NAD + NADH
    14. 14. Activity of MfdC +ATP and DNA Time (minutes) Absorbance at 340nm ATP Avg. = 140 mM ATP/min/ mM enzyme DNA Avg. = 332 mM ATP/min/ mM enzyme - DNA + DNA
    15. 15. Future Work <ul><li>Get repeatable data for crystallization of MfdC with MgCl 2 and AMPpnp </li></ul><ul><li>Proceed with crystallization of MfdC with MgCl 2 , AMPpnp, and 26-mer dsDNA </li></ul>