Post on 14-Jul-2015
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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