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![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 ,[object Object],[object Object],Images courtesy of Karsten Theis](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)
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05.14.10 Theis Lab Group Presentation
- 4. Sequence and structure of Mfd Darst and coworkers (2006) PDB ID 2EYQ Images courtesy of Karsten Theis F632 R905
- 5. R905 Reaches around to aid in ATP hydrolysis Images courtesy of Karsten Theis Phe632 Gln605 Lys634 WalkerB WalkerA apo-Mfd Val454
- 7. Minor groove binding via phosphate-Arg interaction PDB ID 2EZD Images courtesy of Karsten Theis VPTPKRPRGRPKGSKNKGG
- 12. NADH Assay MfdC ATP ADP+P i Pyruvate Kinase Phosphoenolpyruvate Pyruvate Lactate Dehydrogenase Lactate NAD + NADH
Editor's Notes
- - Genes being actively transcribed are preferentially repaired by cells -Persistence of un-repaired DNA damage can cause mutations and other detrimental factors that can lead to cancer, cell death, aging, and Cockayne’s syndrome - Cockayne’s syndrome is a rare autosomal recessive congenital disorder characterized by growth failure, impaired development of the nervous system, abnormal sensitivity to sunlight, and premature aging. - Hearing loss and eye abnormalities are also common, but problems with all internal organs are possible.
- - When RNA polymerase attempts to transcribe damaged DNA, it will sometimes get stalled on pyrimidine dimers - Mfd then recognizes the stalled RNA polymerase, then, the C-terminal region binds to the DNA behind (upstream of) RNA polymerase. - MfdC uses its ATP motor to cause MfdC and MfdN to open up - MfdN moves to sort of push RNA polymerase off of the DNA - Mfd somehow recruits UvrA which forms a dimer on the damaged stretch of DNA, UvrB gets recruited - At this point UvrC an endonuclease excises a piece of DNA equal in length to its active sites flanking the damaged DNA, then DNA polymerase and ligase come in to fill the gap
- -Mfd is made up of MfdN and MfdC which are tethered to each other by a linker between them -There is MfdN, which is composed of domains 1 through 3. Domain 2 binds UvrA and UvrA binding is inhibited by domain 7. -There is MfdC which is composed of domains 4 through 7. Domain 4 binds RNAP, domain 5 binds ATP, we don’t know where DNA binds. - The R905 residue is important for ATP hydrolysis
- - The role of F632 is being investigated by Dave - For ATP hydrolysis in MfdC to happen, the R905 residue depicted in the crystal structure 1 slide back, swings around to neutralize negative charge on ATP after DNA binds - Mg2+, and K634 also help stabilize the negative charge on the ATP - The D&E residues deprotonate a water moleculue and OH- attacks the ATP displacing the gamma phosphate
- -I work with the ATHook-MfdC. It has a small polypeptide addition attached to the N-terminus of Domain 4 of MfdC. It tends to bind well to 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 - The specificity for AT-rich regions comes from _______
- - The ATHook portion of MfdC makes contacts with DNA on the phosphate backbone of the minor groove with 3R and 2K residues via H-bonding the phosphate backbone
- - In the past, I have had issues with concentration and yield. I was working with the Iq cell line. I decided to do a fresh transformation into LysY Iq to try to sidestep the yield and concentration issues I had been having - I also going to try reversibly precipitating the protein with ammonium sulfate or PEG-4000, then checking for success or failure with an NADH assay
- - Most of the MfdC bound to the Ni Column and a little came out in the wash step - The fractions are really impure because I only have a 10ml wash on a 5ml column - NiE2 precipitated as it was being eluted from the column which I’m guessing is the smear in NiE2. The elution fraction was completely opaque but then NiE3 was clear again
- - I spun down the NiE2 fraction at 14,000 rpm for 15 minutes and a lot of the protein pelleted out in the tubes - I saved the supernatant and loaded 10ul into the gel on the previous slide labeled as NiE2 sup - Then I desalted the supernatant, and pooled it with the rest of my desalted fractions - I added 400ul of Lysis buffer to each of the tubes and tried to get some of the pellet to redissolve then loaded 10ul in the last lane of the gel on the next page. I have it labeled as NiE2 precip
- -The precipitate was mostly MfdC. Judging by band width comparison, it appears slightly less intense than HepE2 which was at about 3mg/Ml. And that was just the pelleted protein that went back into solution - Back to the purification. The desalts were diluted from 500mM NaCl to 250mM with 10mM Tris and then loaded onto a Q column. MfdC has a net negative charge and so we expected most of it to be in the flowthrough. The column was washed with Lysis buffer. The Q column got rid of most of the contaminating 55ish kd bands as well as most of one below 72. It also helped to get rid of some of the low MW bands, but I took a small hit on yield in the wash fraction - The QFT was then loaded onto a 1mL Heparin column which mimics the phosphate background of the DNA minor groove. The protein was already in 250mM salt and was eluted with lysis buffer - QFT maybe around .6mg/mL -Total Estimated yield=5.5mg + precipitate Hep E1=.1mg/mL Hep E2=2.94mg/mL Hep E3=1.07mg/mL Hep E4=.561mg/mL Hep E5=.441mg/mL Hep E6= no read Hep E7= no read
- I ran an NADH assay to determine if the protein was active This assay uses the fast enzymes Pyruvate Kinase and Lactate dehydrogenase to measure the rate of ATP hydrolysis by measuring the rate of disappearance of NADH at 340nm I was planning on using this assay this assay to determine whether or not the method of Ammonium sulfate precipitation or PEG-4000 precipitation worked and whether MfdC was able to retain its activity after treatment
- I used HepE5 for this assay. [Protein] was .441mg/mL but I diluted it by 5 to a [stock] of .0882mg/mL for this assay The three runs had an average specific activity of about 9mM ATP/min/ mM Enzyme with background subtracted, again, normal activity is between 80 and 120 This activity is far lower than the expected value probably due to the precipitation that occurred while eluting from the Nickel column in E2 so it didn’t make sense to try reversable precipitation