AFM visualization of MutS Sliding Clamp Formation in DNA Mismatch Repair

Erie GroupZimeng Li2012-5-30AFM visualization of MutS Sliding Clamp Formation in DNA Mismatch RepairContentsDNA Mismatch RepairSliding Clamp FormationAFM techniques in fluidResultsFuture To-dos

DNA Mismatch Repair

Could excise at either 3nick and 5nick?Whats the stop signal?Does sliding clamp consumed energy?primers at the replication fork may provide the strand discrimination signal

MMR in E. coli is initiated when MutS (Table 1) binds to mismatched DNA. MutS interacts with the -clamp accessory protein (64) that is required for pro-cessive DNA replication, and it may help deliver MutS to mismatches. Correction of the replication error requires that the newly synthesized strand be targeted for excision. This process is accomplished in an ATP-dependent manner when MutS interaction with MutL activates the latent endonuclease activity of MutH. MutH is a member of the type II family of restriction endonucleases whose crystal structure has been described (65). It cleaves the newly synthesized, temporarily unmethy-lated strand at hemimethylated GATC sites located within about 1kb of the error. The resulting nick, which can be either 3 or 5 to the mismatch, is the entry point for MutL-dependent loading of DNA helicase II and binding of single-strand DNA-binding protein. Working together, these proteins generate single-stranded DNA (ssDNA) that is digested by either 3 or 5 exonucleases, depending on the location of the nick relative to the mismatch. This excision removes the error and allows highly accurate DNA polymerase III to correctly resynthesize the strand. DNA ligase seals the nick to complete MMR.

3Sliding Clamp Formation

Tessmer, et.al (2008)A - smooth angle kink at Non-specific sites; B - Glu recognize MM site and form H bond to it; C - Phe stacks with MM bases on the DNA; D - ATP uptake inducing conformational change to form mobile clamp. Note: URC once formed, combined with ATP activation/uptake, makes URC irreversible. Thus, URC only need to be populated slightly to signal repair

4Sliding Clamp Formation

Qiu, et.al (2012)D:DOur results imply that this transition does not occur when MutS is doubly liganded with ADP. Notably,although mismatched DNA stimulates release of ADP, the rate of ADP release is similar to or slightly slower than the rate of ATP-induced release of MutS from unblocked DNA, and it is four times slower than the rate of release of doubly ADP-liganded MutS from the mismatch observed here. These results suggest that the doubly liganded ADP MutS is not converted to a sliding clamp in the presence of ATP. D:TSteady-state measurements in the presence of nucleotides suggest that both ATP and ADP must be bound to MutS during its conversion to a sliding clamp form that signals repair.Domain movementUpon binding homoduplex DNA, MutS predominantly adopts a high FRET state in which domains I are closed and then converts to a low FRET state in which domains I are open, while still on DNA, in both the presence and absence of ADP and ATP.

5Myosin V walk/Nano robot spider walkSuper resolution microscopy (Yildiz(2003);Nils Walter)AFM (Kodera(2010);Nils Walter)FRET (Qiu(2012))?TechniquesJake whats your bp sem that deal with this model?6Atomic Force Microscopy

From Force to Distance

Two basic modes contact and ac, dual AC, like EFMWhy not use contact

8AFM

Interaction of tip with sample is like to springs why spring constant is important

9AFM

Q factorWhy repulsive shift the res freq use spring model to define thisHow to choose repulsive

10Fluid AFM techniquesDifference between thermal tune and cantilever tuneSubstrates, special tips/treatmentAtomic Force MicroscopyResults

Recall

DNASample BufferAPTES?Rinse?In Air?Image BufferTip

NiCl2No APTES, No Rinse, No Air DryActivated charcoal15

Buffer filter (twice)Water - filterCantilever holder Contamination

RMS: 220pmRMS: 37pmRoot mean square16Substrate/Support

New Substrate Construct

RMS: 220pmRMS: 80 pm

Dirty Tips?

RMS: 80 pmRMS: 500 pmDNA: 600pm, Background: 400pm, Dots: 6nmFinally Solved Contamination

WaterLoSHiSNoSDryFluidDryFluidDryFluidDryFluidDecent Image29%(6/21)18%(2/11)50%(1/2)100%(1/1)Great Image38%(8/21)18%(2/11)13%(1/8)Total67%36%50%13%0%(1)100%Success Probability

Tip consumption: ~70, 30% gets DNA, 30% gets something real, 30% badRinse twise result in no dna 21Resolution compareFast image capabilityBuffer dependenceInjectionEvaluate operationsCase Study

Resolution Improvement?

AirWater23

Tip is hit so easily

HeightPhaseGot contaminated easily24Fast Imaging CapabilityScan speed, scan points, integral gain, scan size; most importantly, tips health12s, 25s,50s512, 2512,4256,6

256, 6256,10256,3

96,396,10200,10200,20512,20

Conclusion: 150nm scan10s: 100,1020s: 200,10512,401024,20512,20100,10

Conclusion: 300nm scan25s: 512,2010s:100,10

512,20256,20256,40Conclusion: 100nm scan12s: 256,206s: 256,40

One of the dna is moving29

256,5256,1096,10512,10512/256,20Conclusion: 1um scan50s:512,10;256,525s:256,10

One of the dna is moving30

Old Tips avoid itTips are consumable, costly, experiment is expensive31Injection

As time goes up, dirty things come out

10:11:33pm8:09:06pmInjection of MutS

BeforeAfterDidnt see protein dirty/low c/water instead of buffer34How is DNA bound to mica?Competition between Mg2+ and Na+Working with salt bufferHi-salt buffer

Direct fluid imaging, 512,2.3

512,5Cannnot scan fast dependent on tipsShould have been using low scan points37Indirect fluid imaging (i.e. dry first)Initial: 120uL water+DNAInjection: 120uL Hi-salt ->lo-salt mixtureEvaporate: 120uL waterEventually: 120uL Hi-salt+DNA

Hi-salt buffer

Hi-salt buffer injection

BeforeAfter

After evaporation

BeforeAfter

After evaporation

After evaporation

Dna Doesnt move42

Lo-salt Buffer

9:45:20pm9:55:27pmIndirect/direct fluid image: doesnt move43WaterLoSHiSNoSDryFluidDryFluidDryFluidDryFluidDecent Image29%(6/21)18%(2/11)50%(1/2)100%(1/1)Great Image38%(8/21)18%(2/11)13%(1/8)Total67%36%50%13%0%(1)100%Success Probability

Tip consumption: ~70, 30% gets DNA, 30% gets something real, 30% badRinse twise result in no dna Guess low success rate caused by free dna in the fluid, esp cdna that traps tips. Linear dna may be better44Great imageDecent imageNew tips31Old tips12My operations wrong?

100% success4515minRunning a full cleaning cycleDeposition/No incubationRinse once (Twice results in no DNA?)15-30min/cycleImage/No Good?/Change tips/Image/

Average 2~3tips/sample

Current Protocol2h per exp that sees something good luck; 6hour/12hour bad luck46Continue working with lo-salt bufferIf not working, try no-salt bufferAdding NaCl to increase DNA mobilityHi-salt using APTES treated mica to reduce mobilityDoes MutS work in water or no-salt buffer?To-dosWorks in Nicl and should work with mgcl. 47