12.18.09 theis lab group presentation

15
Biochemistry and Molecular Biology Chris Sandifer Crystallization and ATPase Activity of MfdC Theis Lab

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Biochemistry and Molecular Biology

Chris Sandifer

Crystallization and ATPase Activity of MfdC

Theis Lab

2Biochemistry and Molecular Biology

DNA Damage

DNA Repair

Mutations

Replication ErrorsPersistent DNA DamageGenomic Instability

Cancer

Cell deathAging

Replication

Transcription and DNA repair Transcribed genes are preferentially repaired Defect in transcription-coupled repair in humans

leads to Cockayne’s syndrome

DNA damage, cancer, aging

Images courtesy of Karsten Theis

3Biochemistry and Molecular Biology

Sequence and structure of Mfd

Darst and coworkers (2006)PDB ID 2EYQ

Images courtesy of Karsten Theis

4Biochemistry and Molecular Biology

Mfd, the bacterial TRCFRNA polymerase stalled at DNA

damage is recognized by Mfd

UvrB

pre-incision complex (UvrB “padlock” bound

to damaged DNA) RNA,RNAP Mfd UvrA2

UvrA2 UvrB

RNA PolMfd

C

N

COUPLING

Removal of RNA polymerase• Make DNA damage accessible• Rescue arrested transcription (transcription regulation)

Recruitment of DNA repair enzymes• Has to be faster than next polymerase arriving at site

Images courtesy of Karsten Theis

5Biochemistry and Molecular Biology

Goal: Obtain crystal structures of MfdC:ATP and MfdC:ATP + DNA

7

54

6

DNA groove

Proposed path of DNA •Optimal DNA length

for crystallization

•Identify DNA-binding residues

•Conformational changes

6Biochemistry and Molecular Biology

My MfdC Mutant

Low ATPase activity (turnover ~10/min)

No robust DNA binding• Need to add non-hydrolysable

ATP analogs• Need to use DNA of >100 bp

ATPase activity hardly stimulated by DNA (1.2-fold)

No ATP binding site available in apo conformation

Full Length Mfd: ATHook-MfdC: High ATPase activity (observed

turnover ~100 to 150 mM ATP/min/mM Enzyme)

ATHook binds DNA strongly• Binds to AT rich areas of DNA

very well• Can use DNA of < 100 bp

ATPase activity substantially stimulated by DNA (~2.0-fold)

7Biochemistry and Molecular Biology

Grow Up & Purification of MfdC

T7 Iq Cells with ATHook-MfdC Incubate at 37°C, Induced at 30°C Ni Affinity Column Desalt Anion Exchange Column Heparin Affinity Column Sizing Column 1mL Heparin Affinity Column (To concentrate)

8Biochemistry and Molecular Biology

SDS-PAGE Nickel and Anion Exchange 4L Grow Up

NiFT NiW NiE1 QE1QFT4NiE4NiE3NiE2 Mark QFT2 QE3 QE4QE2

80

9Biochemistry and Molecular Biology

First Heparin Column

80

10Biochemistry and Molecular Biology

Sizing Column

80

38

11Biochemistry and Molecular Biology

Final Heparin Column

Final Heparin Column

Mark

80

HepFT HepW HepE7HepE6HepE5HepE4HepE3HepE2HepE1

12Biochemistry and Molecular Biology

Crystallization of MfdC with MgCl2 and AMPpnp

Concentrated in drop with volatile buffer, Ammonium Acetate .5M

1:6 Ratio of Precipitant to protein

Crystal formed in .2M Sodium Acetate Trihydrate, .1M Sodium Cacodylate pH 6.5, 3% w/v PEG 8000

~20μM

13Biochemistry and Molecular Biology

ATPase Assay

MfdC

ATP ADP+Pi

Pyruvate Kinase

PhosphoenolpyruvatePyruvate

Lactate Dehydrogenase

Lactate

NAD+ NADH

14Biochemistry and Molecular Biology

Activity of MfdC +ATP and DNA

Time (minutes)

Ab

sorb

ance

at 3

40n

m

ATP Avg. = 140 mM ATP/min/ mM enzyme

DNA Avg. = 332 mM ATP/min/ mM enzyme - DNA

+ DNA

15Biochemistry and Molecular Biology

Future Work

Get repeatable data for crystallization of MfdC with MgCl2 and AMPpnp

Proceed with crystallization of MfdC with MgCl2, AMPpnp, and 26-mer dsDNA