Biochemistry and Molecular Biology

Chris Sandifer

MfdC Purification and Concentration

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

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

4Biochemistry and Molecular Biology

Sequence and structure of Mfd

Darst and coworkers (2006)PDB ID 2EYQ

Images courtesy of Karsten Theis

F632R905

5Biochemistry and Molecular Biology

R905 Reaches around to aid in ATP hydrolysis

Phe632

Gln605

Lys634

WalkerB

WalkerA

apo-Mfd

Val454

Images courtesy of Karsten Theis

6Biochemistry and Molecular Biology

The ATHook-MfdC Mutant

ATHook inserted at N-terminus of Domain 4 (VPTPKRPRGRPKGSKNKGG)

High ATPase activity (observed turnover ~80 to 120 mM ATP/min/mM Enzyme)

ATHook binds strongly to the phosphate background of the DNA minor groove• 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

Minor groove binding via phosphate-Arg interaction

VPTPKRPRGRPKGSKNKGG

PDB ID 2EZD

Images courtesy of Karsten Theis

8Biochemistry and Molecular Biology

Objectives

Find a way to increase concentration by:• Reversibly precipitating MfdC with Ammonium Sulfate• Reversibly precipitating MfdC with PEG-4000

Run NADH assays to determine if precipitation affects specific activity

Crystallize MfdC with AMPpnp and Mg2+

9Biochemistry and Molecular Biology

Ni2+ Affinity Column (5mL)

5hr induction at 30°C 200mL lysate 10mL Wash (20mM

Imidazole) Elutions are 4mL into

1mL lysis buffer• Lysis Buffer: .5M NaCl, 10mM

Tris pH 8.2• Wash Buffer: .5M NaCl, 50mM

Tris pH 8.2, 20mM Imidazole• Elution Buffer: .5M NaCl,

10mM Tris pH 8.2, 250mM Imidazole

10Biochemistry and Molecular Biology

NiE2 Precipitation

NiE2 had a lot of aggregate

Spun down at 14,000rpm for 15minutes

Desalted Supernatant Added 400ul lysis buffer to

pellet SDS sample is last lane on

next gel

11Biochemistry and Molecular Biology

QPool = 26mL QFT= 26mL QWash = 10mL HepFT= 26mL HepW= 10mL HepE1-E7=

1mL each

Binding Buffer: 250mM NaCl, 10mM Tris pH 8.2

Anion Exchange (1mL) and Heparin Affinity (1mL)

12Biochemistry and Molecular Biology

NADH Assay

MfdC

ATP ADP+Pi

Pyruvate Kinase

PhosphoenolpyruvatePyruvate

Lactate Dehydrogenase

Lactate

NAD+ NADH

13Biochemistry and Molecular Biology

The ATPase Activity of MfdC

Ab

sorb

ance

at

340

nm

Time (minutes)

•Average Specific activity: 10mM ATP/min/mM Enzyme

• 9mM ATP/min/mM Enzyme with background subtracted

14Biochemistry and Molecular Biology

Future Work

2L grow-up of T7 Lys Y Iq induce for 3hrs Attempt to concentrate if needed with:

• 2M Ammonium Sulfate• 50% PEG-4000

Crystallize MfdC with AMPpnp and Mg2+