05.14.10 Theis Lab Group Presentation
Transcript of 05.14.10 Theis Lab Group Presentation
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