major qualifying project - worcester polytechnic institute · 2017-04-27 · mapping dna...
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Major Qualifying Project
MAPPINGDNAREPLICATIONINTHEYEASTSPECIES
SACCHAROMYCESCEREVISIAE
AnnaWortman
Date:4/27/2017
MAPPINGDNAREPLICATIONINTHEYEASTSPECIESSACCHAROMYCES
CEREVISIAE
AMAJORQUALIFYINGPROJECTSUBMITTEDTOTHEFACULTYOFWORCESTERPOLYTECHNICINSTITUTEINPARTIAL
FULFILLMENTOFTHEREQUIREMENTSFORTHEDEGREEOFBACHELOROFSCIENCE
SubmittedBy:
AnnaWortman
Submittedto:
Advisor:ProfessorReetaRao
Date:4/27/2017
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ABSTRACT DNAreplicationisanessentialfunctionthatallowsalllivingorganismstomaintainlife.
This function is coordinated with other aspects of genomemetabolism such as DNA repair,
chromatinstructure,andgeneexpression.Timingisimportantinthiscoordination-thetiming
ofreplicationoriginfiringsdrivesreplicationtiming.Onewaytimingisregulatedisbyloadingof
thereplicativehelicasecomplex,MCM.AnoriginismorelikelytofireearlierwhenmoreMCM,
ahexamerofsixpolypeptidesthataidsintheformationandelongationofthereplicationfork,
isloadedbytheOriginRecognitionComplex(ORC).
The goal of thisMQP is to examine thenumberofMCMcomplexes loadedon single
replicationoriginsbeforeSphase,thesynthesisphaseofDNAreplication,isinitiated.Inorder
to identify the specific number of MCM complexes that are loaded on any given origin, a
SaccharomycescerevisiaestrainwasbuiltthatcontainsaTALO8plasmidwithasingleorigin,a
LacI-SNAPcassettetotetherTALO8,andaMCM4-fluorescencecassettetomicroscopicallytrack
MCM.Thisplasmidwillbecapturedinaflowcellandmicroscopicallystudiedinordertocount
thenumberofMCMsloadedonsingleplasmidscontainingknownorigins.
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ACKNOWLEDGEMENT
IwouldliketothankDr.NickRhindforgivingmetheopportunitiestovolunteerinhis
labandinvitingmetodothisresearchproject.Hissupport,guidance,andadvicehavehelped
me immensely throughout thisproject. Iwouldalso like to thankLivioDukaj forassistingme
throughout the laboratory procedures and taking the time to answer all of my questions, I
couldn’t have completed this project without him. I’d like to extend thanks to my advisor,
ProfessorReetaRao, for guidingme through thisprocess. Finally, Iwould like toexpressmy
gratitudeforthesupportfrommyfamilyandfriends.Withoutalloftheirhelp,Iwouldnothave
beenabletocomesofarinmyacademicjourney.
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TABLEOFCONTENTS
Abstract...................................................................................................................................i
Acknowledgement..................................................................................................................ii
TableofContents..................................................................................................................iii
TableofFigures.....................................................................................................................iv
ListofTables...........................................................................................................................v
Introduction...........................................................................................................................1
ReplicationTiming..............................................................................................................1
StochasticModelofReplication..........................................................................................3
StochasticModelEvidence..................................................................................................3
HypothesesoftheStochasticModel...................................................................................7
SingleMoleculeBiochemistry.............................................................................................8
MaterialsandMethods.........................................................................................................12
Strains,Plasmids,andPrimers..........................................................................................14
VerificationofStrainsandQualityControl........................................................................14
PlasmidMiniprep..............................................................................................................14
YeastTransformation........................................................................................................15
BacterialTransformation..................................................................................................15
NoodleMaking.................................................................................................................16
BallMillGrindingofYeastNoodles...................................................................................16
Results..................................................................................................................................18
StrainEngineering.............................................................................................................18
Discussion.............................................................................................................................21
Bibliography.........................................................................................................................23
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TABLEOFFIGURESFigure1.RibbondiagramshowingthetopandsideviewsofahexamermodelofMCM
ReprintedfromBrewsteret.al.(2008)……………………………………………………………………………………….2
Figure2.MCMChIP-seqdata.ReprintedfromDaset.al.(2015)……………………………………………….5
Figure3.ARS1hasmultipleMCMcomplexesloadedinvivo.ReprintedfromDaset.al.(2015)…6
Figure4.PossibleMCMdistributionsataknownearlyorigin-ARS1,withanaverageofabout3
MCMs.A)Poissondistribution,expectedifMCMloadingbasedonrateofloadingbyORC.B)
Saturationmodel,expectedifMCMloadingbasedoncapacityforMCMs…………………………………8
Figure5.ImagingwithSNAP-tagTechnology:ClonegeneofinterestintoNEBexpressionvector.
2)Transfectplasmidfusionintocells,proteinisexpressedincells.3)Addlabelofinterest.4)
Covalentmodificationoccurs,labelingproteinforvisualization.Reprintedfrom(SNAP-tag
Technologies:NovelToolstoStudyProteinFunction)……………………………………………………………….9
Figure6.SinglemoleculepulldowntoquantifyMCMcomplexes……………………………………………11
Figure7.yFS989strainwithMCM4-GFP………………………………………………………………………………….18
Figure8.Plasmidconstructionprocessandconfirmation………………………………………………………..20
Figure9.Summaryoftransformation………………………………………………………………………………………20
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LISTOFTABLESTable1:Strainsandplasmidsusedinthisproject…………………………………………………………………….12
TableTwo:Primersusedinthisproject……………………………………………………………………………………13
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INTRODUCTION
Replicationtiming
DNAreplication ineukaryoticcells is limitedtoaspecificwindowof time,knownasS
phase.Toadvancesuccessfullythroughthisphase,theentiregenomemustbecopiedcorrectly,
exactlyonce,andwithinatimelymanner;otherwise,errorsinreplicationcouldleadtogenome
instabilityandcelldeath(Bell&Kaguni,2013)(Rhind&Gilbert,DNAreplicationtiming,2013).
Tothisend,DNAreplicationhasevolvedtobeapreciselycoordinatedprocess,dependenton
anorderedseriesofsteps inordertoproducethefactorsnecessary forallphasesof thecell
cycle.
The process of eukaryotic replication begins from several locations, or origins of
replication,oneachchromosome.Thetimingofreplicationoriginfiringdeterminesreplication
timing(Goren&Cedar,2003).OccurringduringtheG1phaseofthecellcycle,replicationorigin
loadingbeginswhentheOriginRecognitionComplex,orORC,bindstothegenome’soriginsand
loads the eukaryotic replicative helicase complex, minichromosome maintenance complex
(MCM),aroundtheDNA(Sakakibara,Kelman,&Kelman,2009)(Yeeles,Deegan,Janska,Early,
&Diffley,2015).
MCMproteinismadeupofabout650aminoacidsstructurallydividedintothreeparts:
anN-terminal,acatalyticregion,andaC-terminalhelix-turn-helixdomain(Sakakibara,Kelman,
&Kelman,2009).AheterohexamericMCM2-7helicasecomplexformsatreplicationoriginsto
unwinddoublestrandedDNAandpowerforkprogression(Figure1)(Brewster,etal.,2008).
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Figure1.RibbondiagramshowingthetopandsideviewsofahexamermodelofMCM.ReprintedfromBrewster
et.al.(2008)
OncetheMCMcomplexisactivated,replicationisinducedandreplicationtimingforthe
genome is determined.Within this phase, someportions of the genome replicate early, and
others late, creating a characteristic pattern of replication timing. Recent models propose
stochastic regulation of origin firing wherein firing time of an origin within a population is
equivalent to the probability of that origin firing at the single-cell level (Yang, Rhind, &
Bechhoefer,2010)(Das,Borrman,Liu,Bechhoefer,&Rhind,2015).Anoriginthatfireswithhigh
probabilityismorelikelytofireearlyinSphasewhileanoriginthatfireswithlowerprobability
is unlikely to fire early in S phase,will have a later average firing time andwill be passively
replicated(Yang,Rhind,&Bechhoefer,2010)(Das,Borrman,Liu,Bechhoefer,&Rhind,2015).
As cells differentiate, origin firing patterns change and correspond to patterns of
transcriptional regulation and chromosome structure, implying a relationship between
replication timing and processes of genome metabolism like gene expression and genome
evolution (Goren & Cedar, 2003) (Rhind & Gilbert, DNA replication timing, 2013). However,
thereareuncertaintiesintheregulationoforiginfiring,leadingtotwodifferenthypothesesfor
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the stochastic model (Rhind, Yang, & Bechhoefer, Reconciling stochastic origin firing with
definedreplicationtiming,2010).
StochasticModelofReplication
Thestochasticmodelofreplicationsuggeststhatthefiringtimeofanindividualoriginof
replication inapopulation isheterogeneous, thismodelassumesthat the firingofoneorigin
does not affect another and that the process is independent (Bechhoefer & Rhind, 2012).
Studies have demonstrated heterogeneous patterns of origin firing (Patel, Arcangioli, Baker,
Bensimon, & Rhind, 2006). From them, two central conclusions have been drawn- first, the
firingofeukaryoticreplicationoriginfiringisinefficientandsecond,stochastic.Theinefficiency
ofeukaryoticorigins iswelldocumented(Kalejta&Hamlin,1996)(Czajkowsky,Liu,Hamlin,&
Shao,2008).Someyeastoriginsareashighas90%efficientandotherslessthan10%(Hiraga,et
al., 2014). This inefficiency suggests some origins are passively replicated by a fork from
anothernearbyorigin; thus, the longeranorigingoeswithoutbeingpassively replicated, the
betterchanceithasoffiringindependently.
StochasticModelEvidence
Experimentally,thestochasticmodelhasbeenaddressed ina largepopulationofcells
basedonmathematicalanalysisofreplicationkinetics.Thismodel,basedonthebuddingyeast
Saccharomyces cerevisiae, outlines a multiple-MCM system in which replication timing is
dependentonthenumberofMCMcomplexesloadedonoriginsofreplication(Yang,Rhind,&
Bechhoefer,2010)(Rhind,Yang,&Bechhoefer,Reconcilingstochasticoriginfiringwithdefined
replication timing, 2010). Following this model,MCM complexes are stochastic and activate
withsimilarprobabilitiesacrossthegenome.OriginswithmoreMCMcomplexesloadedare,on
average,morelikelytofireearlyinSphase(Das,Borrman,Liu,Bechhoefer,&Rhind,2015).
The multipleMCMmodel was first used to show that early-firing origins have more
MCM complexes loaded than do later-firing origins. Using ChIP-seq in G1 arrested cells, the
genome-widedistributionofMCMcomplexeswasexamined.Thesignalwasconcentratedon
known origins,with increased levels on those that fire earlier: ARS1012, ARS1014, ARS1018,
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andARS1019(Fig.2A).Thisdatashowedthatthere isastrongcorrelationbetweentheChIP-
seq signal and the origin timing parameter, n, across the genome (Fig. 2B) (Yang, Rhind, &
Bechhoefer,2010).Nisadirectestimateoforigintiming,describingthefiring-timedistribution
oforiginsandnotoriginreplication,which is influencedbypassivereplication(Das,Borrman,
Liu, Bechhoefer,&Rhind, 2015). The originswhose signal falls above the diagonal represent
moreMCMcomplexesloadedthanthemathematicalmodelpredicted.Theseincludetelomeric
origins, which are known to fire late in a hetero- chromatin-dependent manner and those
delayedinfiringbyRpd3HDAC,ahistonedeacetylasethatremoveslysineresiduesontheN-
terminalpartofthecorehistonesandpreventstranscription(Das,Borrman,Liu,Bechhoefer,&
Rhind,2015)(UniProtKB-P32561(RPD3_YEAST)).
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Figure2.MCMChIP-seqdata.ReprintedfromDaset.al.(2015)
This data was used to conclude that the relative number ofMCM complexes loaded
during G1 regulates origin firing timing during S phase based on results that only used the
relativenumberofMCMsatorigins(Das,Borrman,Liu,Bechhoefer,&Rhind,2015).
This model was next used to illustrate that early-firing origins have multiple MCM
complexes loaded. Different origins were engineered into the TALO8 plasmid system and a
singlebinding site for the zinc-fingerDNAbindingproteinZif268was introduced.MCM2and
ORC2weretaggedwiththeHAepitopeandexpressedanHA-taggedZif268(Das,Borrman,Liu,
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Bechhoefer,&Rhind,2015).TheTALO8plasmidscontainingdifferentoriginswerepurifiedand
Westernblottingwasused todeterminehowmanyMCMcomplexeswere loadedrelative to
theZif268control(Fig.3A,B).AfterprovidingevidencethatmultipleMCMscanbeloadedona
singleoriginandaffectfiringtime,thismodelwasusedtoshowthatreducingthenumberof
MCM complexes loaded, delayed the firing time of the affected origin (Das, Borrman, Liu,
Bechhoefer,&Rhind,2015).
Figure3.ARS1hasmultipleMCMcomplexesloadedinvivo.ReprintedfromDaset.al.(2015)
The evidence from thismodel presents amechanistic overview of how replication is
timed and regulated in Saccharomyces cerevisiae (Das, Borrman, Liu, Bechhoefer, & Rhind,
2015). It supports themodel of replication timing as a stochastic eventwith competition at
origins for rate-limiting activators. The experimental data suggests that origins that compete
moreefficientlyforlimitingactivatorsaremorelikelytofireearly,andthus,replicateearly.The
numberofMCMcomplexesloadedatoriginscontributestothiscompetitionandleadstothe
efficiency and timingof origin firingduring S phase.OneMCM is nomore likely to fire than
another, but increasing the number ofMCM complexes at an origin increases the likelihood
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thatitwillfireearlierthananoriginwithfewercomplexes.Therefore,thismodeldemonstrates
a “biochemically plausible mechanism for regulating origin efficiency and timing” based on
experimentation with a large population of cells (Das, Borrman, Liu, Bechhoefer, & Rhind,
2015).
HypothesesoftheStochasticModel
Although the stochastic model illustrates how replication timing is regulated in a
population array, it leads to important questions about howMCM complexes are loaded at
singleorigins.Asstatedpreviously,therearetwohypothesesforwhichMCMloadingmaybe
regulated:therateatwhichMCMsareloadedorthecapacityforMCMloading(Yang,Rhind,&
Bechhoefer, 2010). If MCM loading is rate dependent due to the specific activity of ORC
determining howmany complexes are loaded,with no effect from crowding, then a Poisson
distributionofMCMnumbersisexpected(Birnbaum,1954).Askewedbell-shapedcurvewitha
rightwardtailwouldbeexpected(Figure4A)(Das&Rhind,2016).However,ifthecapacityfor
MCMcomplexes tobe loaded is limited,withhigh capacityorigins able to loadmoreMCMs
than low capacity origins, a saturationmodel would be expected. ORCwould load asmany
MCMs as will fit at an origin, but then add additional MCMsmore slowly (Figure 4B) (Das,
Borrman,Liu,Bechhoefer,&Rhind,2015).
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Figure4.PossibleMCMdistributionsataknownearlyorigin-ARS1,withanaverageofabout3MCMs.
A) Poissondistribution,expectedifMCMloadingbasedonrateofloadingbyORC.B) Saturationmodel,expectedifMCMloadingbasedoncapacityforMCMs.
SingleMoleculeBiochemistry
Previously citedbiochemicalanalysisof replication timingprovided themultipleMCM
modelandthestochastichypotheses.However,thedatawaslimitedinthatitonlyestimated
theaveragenumberofMCMsloadedatorigin(Das,Borrman,Liu,Bechhoefer,&Rhind,2015).
Singlemoleculebiochemistry canbe implementedwith fluorescent, single-molecule counting
of MCMs loaded in vivo to measure the distribution of the number of MCMs loaded on
individualorigins(Friedman&Gelles,2015).
To confirm theMCM complexmodel on a singlemolecule level, specific biochemical
approachescanbeimplemented,includingoriginisolationbyutilizingtheinteractionbetween
thelacoperator(lacO)andthelacrepressorprotein,LacI(Forde,Ghose,Slater,Hine,Darby,&
Hitchcock,2006).Initsnaturalfunction,thelacrepressoractsthroughahelix-turn-helixmotif
located in itsDNAbindingdomain (Schumacher,Choi, Zalkin,&Brennan,1994). Thedomain
bindsbase-specificallytothemajorgrooveintheoperatorregionwithresiduesofrelatedhinge
alpha helices binding to base contacts in the minor groove (Schumacher, Choi, Zalkin, &
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Brennan,1994).ThisbindingincreasestheaffinityofRNApolymeraseforthepromoterregion,
disallowingfordissociationandpreventingtranscriptionofthemRNAencodingtheLacproteins
(Daber, Stayrook, Rosenberg, & Lewis, 2007). This system may be amplified, with multiple
copiesof lacOstably integrated intoaeukaryoticgenome-developingabindingsitearrayfor
LacI(SingleCellManipulations).
TheyeaststraincontainingtheTALO8plasmidwithlacOandARS1isalsotransformed
withMCM4-GFP-allowingforfluorescentlylabeledMCMcomplexestobeloadedtothesingle
plasmidforstudy.PulldownofthisTALO8plasmidisachievedfirstbyusingaSNAP-tag.SNAP-
tag is a 20 kDamutant of the DNA repair protein O6-alkylguanine-DNA alkyltransferase that
covalentlybindsspecificallyandrapidlywithbenzylguanine(BG),labelingtheSNAP-tagwitha
syntheticprobe(Figure5)(SNAP-tagTechnologies:NovelToolstoStudyProteinFunction).This
taggingsystemhasavarietyofadvantages.First,therateatwhichtheSNAP-tagbindstoBGis
independent of the synthetic probe attached to BG (SNAP-tag Technologies: Novel Tools to
StudyProteinFunction).Next, therearenorestrictionsonexpressionhostwiththeSNAP-tag
system. Finally, the SNAP-tag products are chemically inactive towards other proteins,
eliminatingnonspecificbinding(SNAP-tagTechnologies:NovelToolstoStudyProteinFunction).
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Figure5.ImagingwithSNAP-tagTechnology:ClonegeneofinterestintoNEBexpressionvector.2)Transfectplasmidfusionintocells,proteinisexpressedincells.3)Addlabelofinterest.4)Covalentmodificationoccurs,
labelingproteinforvisualization.Reprintedfrom(SNAP-tagTechnologies:NovelToolstoStudyProteinFunction)
Thenext featureofthepulldownisthesyntheticprobeattachedtoBG.Alongwitha
649-fluorophore for labeling, biotin is attached to BG. When the flow cell is coated in
streptavidin, the well-characterized relationship between biotin and streptavidin pulls the
entire singlemolecule complex down. This interaction is one of the strongest, non-covalent
interactions(Chivers,Koner,Lowe,&Howarth,2011).Thehighbindingaffinityisduetoseveral
chemical interactions. First, there is a complementarity between the binding pocket of
streptavidin and biotin with 8 hydrogen bonds made to residues within the binding site
(DeChancie & Houk, 2007). Next, there is a ‘second shell’ involving hydrogen bonding to
residueswithinthefirstshellandnumerousvanderWaalsforcesmadetothebiotinwhenin
the streptavidin pocket (DeChancie & Houk, 2007). Finally, the affinity between streptavidin
andbiotinisinfluencedby“stabilizationofaflexibleloopconnectingBstrands3and4(L3/4),
whichclosesovertheboundbiotin,actinglikea'lid'overthebindingpocketandcontributing
totheextremelyslowbiotindissociationrate”(DeChancie&Houk,2007).
In implementing theaforementionedbiochemistry, singleplasmidmoleculepulldown
canbeachievedandusedtocountthenumberofMCMsloadedonasingleorigin,thustesting
thehypothesisthatoriginsthatfireearlyinSphasehavemoreMCMsloadedthanoriginsthat
firelateinSphase.ThecompleteoutlineofthisprocessmaybeseeninFigure6.
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Figure6.SinglemoleculepulldowntoquantifyMCMcomplexes
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MATERIALS Table1:StrainsandplasmidsusedinthisprojectStrainNumber DescriptionyFS833 MCM4WildTypeyFS930 MCM4-GFPyFS961 LacI-SNAP(cNAT)yFS977 MCM4-GFPandLacI-SNAPyFS979 MCM4-GFP,LacI-SNAP,andTALO8yFS980 MCM4-mNeonGreenandLacI-SNAPyFS981 MCM4-mNeonGreen,LacI-SNAP,andTALO8yFS989 MCM4-GFPyFS990 MCM4-GFPandLacI-SNAPyFS991 LacI-SNAPMMY1198 SEC3-SNAPpFS270 GFP-HPHCassettepFS449 yeGFP-HPHCassettepFS454 mNeonGreen-KANCassettepFS455 mNeonGreen-HPHCassettepFS458 LacI-FLAGCassettepFS466 SNAPCassette
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TableTwo:PrimersusedinthisprojectPrimerName Sequence(5'-3') Use
MH2r cgcacttaacttcgcatctgTTATTAATTGTTACGCAGGGAATGATTGTAGTAGACAGCA CheckingprimerforGFPintegrationinMCM4
MH7r CGAGGGTGTAAGGAGATCAGTTCGCCTGAATAACCGTGTCggtgacggtgctggtttaattaac CheckingprimerforGFPintegrationinMCM4
KN07 AATCAGCTGTTGCCCGTCTC CheckingprimerforintegrationofSNAP.NAT
KN13 TGGTGAAGGACCCATCCAGTCheckingprimerforintegrationofSNAP.NATdownstreamofLacI
MH08 TTATTAATTGTTACGCAGGGAATGATTGTAGTAGACAGCAtgggcagatgatgtcgagg ToisolateGFP-HPHconstructKN09 CGAGGGTGTAAGGAGATCAGTTCGCCTGAATAACCGTGTCaacagtaaaggagaagaact ToisolateGFP-HPHconstruct
LD200 GTCTTCTGATATCCAGGAAG CheckingprimerinsideMCM4forGFP
LD202 CGTTGCCTCATCAATGCGAG CheckingprimerinsideARS1forpFS408
LD203 CAGTGAGCGCAACGCAATTA CheckingprimerforpFS408LD206 CTCGCATTGATGAGGCAACG CheckingprimerforpFS408
LD207 AGTTCCTCGGTTTGCCAGTT CheckingprimerforpFS408
MH4 CGGCACCGACTTTACCATAGCheckingprimerinMCM4for
GFP
LD223 cggtaatacggttatccacag FwdprimerpFS458.1
LD224 caccgcatagggtaataact RevprimerpFS458.2
LD222 agttattaccctatgcggtggacggtatcgataagcttga FwdprimerpFS458.2
LD227 CATTTCGCAGTCTTTGTCCATCTTTGGTGGAGTACAGGATCC RevprimerpFS458.2LD225 GGATCCTGTACTCCACCAAAGATGGACAAAGACTGCGAAATG FwdprimerpFS466LD226 ctgtggataaccgtattaccgTTAACCCAGCCCAGGCTT RevprimerpFS466
LD215CGAGGGTGTAAGGAGATCAGTTCGCCTGAATAACCGTGTCGGTGCTGGAGCAGGTGCAGGAGCTGGTGCT
aacagtaaaggagaagaactCheckingprimerforMCM4-GFP
21bplinker
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METHODS VerificationofStrainsandQualityControl
First, YFS961,YFS930,andMM1198 single colonieswere streak-platedonYPDmedia,
and the knockout plates: –Trp, -Ura, YES, YES NAT, -His, and –Ade. Also, using YFS961 and
YFS930,liquidculturesweremadebytransferringasinglecolonyinto5mLofYPDandspinning
overnightatroomtemperature.Theknockoutplateswereobservedandtheovernightcultures
wereusedtocheckforfluorescenceviamicroscopy.
GenomicDNAPreparation
YFS961andYFS930underwentgenomicpreparationinordertoserveastemplatesfor
PCR amplification. For this process, these candidates were once again cultured overnight in
liquid YPD. Thenext day, 1.5mlof eachwaspelletedby centrifugation at 20,000 x g for five
minutes.Thepelletswereresuspendedin200ulofcell lysisbufferandimmersedinadryice-
ethanolbathfortwominutes.Bothtubeswerethentransferredtoa95°Cwaterbathforone
minute. This process was repeated and then 200uL of chloroform was added. The
microcentrifuge tubes were centrifuged at 20,000 x g and the supernadent was removed.
Severalethanolwasheswerethenpreformedandthepelletwasdriedatroomtemperaturefor
five minutes. Finally, the genomic DNA was resuspended in 40ul of water. A cleanup was
performed on the genomic DNAwith binding andwash buffer in a column purification that
yielded20ul.ThisDNAwasthenusedinTaqPCRreactionswiththeprimersMH2randMH7rfor
YFS930andKN7andKN13usedwithYFS961.Finally,agelelectrophoresiswith1%agarosegel
wasperformedinordertoverifythecandidates.AfreezerstockofYFS930wasobtainedand
the genomic DNA preparation and PCR was repeated in order to move forward with this
candidate.
PlasmidMiniPrep
Next,theGFP-cassetteplasmid,PFS270,wasobtainedfromafreezerstock,streakedon
anLBCarbplate,andusedtocreateanovernightculture.1.5mLofthebacteriawaspelletedat
6,000xgforoneminuteatroomtemperaturethenresuspendedin200ulofSolutionA(50mM
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TrispH7-8,10mMEDTApH8.0)andhad5ulof10mg/mlRNaseAadded.Aftervortexing,200ul
ofSolutionB(200mMNaOH,1%SDS)wasaddedandthemixtureinverted.300ulofSolutionC
(3MKOAcpH5)wasaddedandthenthemixturewaspelletedbycentrifugationat16,000xg
forfiveminutes.Oncedone,450ulofthesupernatantwastransferredtoanewtube,andthe
DNA was ethanol precipitated by adding 2.5V (1,125ul) of 100% EtOH. After centrifugation,
another ethanol wash was preformed using 70% EtOH and then the pellet was dried and
resuspendedin50ulwater.Finally,theplasmidDNAwascleanedupusingcolumnpurification
andelutingwith20ulofwater.
YeastTransformation
Using PFS270, the GFP-HPH cassette was amplified using PCR techniques with the
primersMH08andKN09.400ulofthePCRproductwascleanedupusingcolumnpurification
anda50mlovernightcultureofYFS961was inoculated.Theovernightculturewasthenspun
downat3,000rpmforthreeminutesatroomtemperature.Thepelletwasresuspendedin5ml
ofsterileTEbufferandspundown.Itwasthenresuspendedin5mlofLiAcMixandspundown.
Then,thepelletwasoncemoreresuspendedin250ulofLiAcMixand100ulwasaliquotedinto
an epindorph tube for one transformation.Next, 10ul of the clean PFS270 PCR productwas
addedtothecellsalongwith10ulof10mg/mlSalmonSpermDNA.700ulofPEGMixwasadded
andthetubewasvortexedandthenincubatedfor30minutesat30°C.Aheatshockwasthen
preformedfor15minutesat42°Candthenspundown, removing thesupernadent.Thecells
wereresuspendedin300ulofTEandthenplatedonYPD.Thenextdaythecellswerereplica
platedontoYPDHPH.
BacteriaTransformation
A bacterial transformation was preformed in order to obtain a different fluorescent
plasmidcandidate,PFS449,thatcontainsaGFPderivative. Inordertodothis,atubeofdh5-
alpha E. coli competent cellswas thawedon ice for 10minutes. 50ul of the cellswere then
pipetted into a microcentrifuge tube and 1ul of diluted plasmid 449 DNA was added. This
mixturewasthenplacedonicefor30minutesandthenheatshockedfor30secondsat42°C.It
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wasplacedback on ice for fiveminutes and then950ul of room temperature LBmediawas
added. The tubewas then spun for one hour at 37°C. Several 10-fold serial dilutions of this
mixturewere then plated on LB Carb and grown overnight at 37°C. The next day, overnight
culturesofsixcandidatecoloniesweremadeandaminiprepwaspreformedinordertoobtain
theplasmidDNA.1uloftheDNAwasrunona1%agarosegelinordertodeterminewhichof
the six candidates contained plasmid DNA. The candidates that contained DNA were then
digestedwithseveral restrictionenzyme:SAP1 inorder to linearize,HPA1tocreateadouble
strandedcut,andBST11tocreateadifferentdoublestrandedcut.Finally,a1%agarosegelof
thedigestedproductswasrun inordertodeterminewhichofthecandidatestrulycontained
PFS449.
Noodle-Making
Yeast‘noodles’oftheexperimentalstrainwerecreatedinordertobegrounddownand
usedasyeastextract.Tostart,2Lofmediawasinoculatedwiththeappropriatestrainfroma
starter culture and grown until an OD600 of 1.2-1.5. The cells were spun down in 500ml
containers at 3000 rpm for 10minutes at 4°C. Thepelletwas resuspended in 25mls ice-cold
dH2Oandallresuspendedyeastwascombinedintoone50mlconicaltube.Thecellswerethen
spundownagainat3000 rpm for5minutesat4°C. The supernadentwasdiscardedand the
cellswerewashedwith50mlsofice-colddH2O,thentheywerespunat3000rpmfor5minutes
at4°C.Next,theyeastpastewastransferredtoa5mlsyringeandexpungedintoanew50ml
conical tube containing 25ml of liquid N2. The excess liquid N2 was removed and the yeast
noodleswerestoredat-80°Cuntiluse.
BallMillGrindingofYeastNoodles
Using theRetschBallMillGrinder,previouslymadeyeastnoodleswereground intoa
yeast extract for use in experimentation. First, the ball mill contained was cooled down by
pouringliquidN2overituntila‘bubbling’effectappeared.Theyeastnoodleswereputintothe
container and the cooling process was repeated. The apparatus was placed into the Retsch
machine,lockedintoplace,andgroundfor1m30sat400rpm.Thecontainerwasplacedback
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into liquidN2, theyeastwasdislodgedfromthesides,andthecoolingprocesswasrepeated.
These steps were repeated until the sample was ground a minimum of 6 times and had a
powderyappearance.
TheseprotocolswererepeatedforstrainsyFS833andyFS989andforplasmidspFS458
andpFS466.
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RESULTSStrainEngineering
AtthebeginningofthisprojectthestrainyFS961wasoriginallyusedastheLacI-SNAP
strainthatMCM4-GFPwastransformedintofrompFS270.Atthesametime,MCM4wastagged
with mNeonGreen from both pFS454 and pFS455 and transformed into yFS961 in order to
comparethestrengthof fluorescencebetweenthetwofluorophores-GFPandmNeonGreen.
The MCM4-GFP and LacI-SNAP strain became yFS977 and the MCM4-mNeonGreen from
pFS454andLacI-SNAP strainbecameyFS980.Next, theTALO8plasmidwas transformed into
yFS977andthestrainwaslabeledyFS979.Finally,TALO8wastransformedintoyFS980andthe
newstrainwasnamedyFS981.
Next,experimentationmovedforwardwithyFS979.Yeastnoodlesweremade,theball-
millgrinderwasusedtomakeyeastextract,theBG-biotin-649biotin-fluorwasconjugated,and
thecomplexwasrunoverflow-cellsfunctionalizedwithbiotinandstreptavidin.
At this point in the project we began the process over due to the presence of a
previously annotated LacI-FLAG present in yFS961 that would compete with LacI-SNAP and
complicate the experimental setup. Starting over began by fluorescently labeling MCM4 by
PCR-based cassette tagging with GFP from pFS270 using the primersMH08 and LD215. The
plasmidpiecewasthentransformedintoyFS833thatcontainedMCM4,thisstrainthenbecame
knownasyFS989(Figure6).
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Figure7.yFS989strainwithMCM4-GFP
To create the LacI-SNAP strain, PCR-based cassette tagging was used to tag a CMV
promoter and LacI from the plasmid pFS458 using the primers LD223 and LD224. Using the
primers LD222 and LD227, the pUC origin, ampicillin resistance marker, and the functional
URA3genewerealsoPCR’d frompFS458.TheSNAP-tagwasPCR’d from theplasmidpFS466
usingtheprimersLD225andLD226.AGibsonAssemblycombinedthethreePCRproducts to
createtheLacI-SNAPplasmidthatwasthentransformedintoE.coli.Theplasmidconstruction
processandconfirmationwithrestrictionenzymeEcoRVcanbeseeninFigure7.Asummaryof
thetransformationprocessisillustratedintheflowchartinFigure8.
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Figure8.Plasmidconstructionprocessandconfirmation Figure9.Summaryoftransformation
TheLacI-SNAPplasmidwasthentransformedintoyPF989tocreatethestrainyFS990.
PCRtaggingofpFS458forLacIandothergenes
PCRtaggingofpFS466forSNAP
GibsonassemblyofPCRproductsandbacterial
transformationintoE.coli
SelectforAmp+coloniesbygrowingcandidatesinLBbroth
withampicillin
Alkalinelysisminipreptoobtainplasmid
SendplasmidoutforsequencinganddigestplasmidwithEcoRVtoconPirmplasmidsequence
DigestplasmidwithBsm1tolinearizeforyeasttransformation
Uncut,EcoRV,Bsm1
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DISCUSSION ThepremiseofthisprojectwastobeabletoquantifythenumberofMCMcomplexes
loaded on plasmids containing single,well-characterized replication origins fromG1 arrested
yeast. This goal was to be accomplished by first producing a yeast strain containing SNAP-
tagged LacI, and MCM4 tagged with GFP. Then, a TALO8 plasmid would be inserted that
containedthewell-characterizedorigin,ARS1,eightcopiesofthelacOhighaffinitybindingsite,
andafunctionaltryptophangeneforselectivepropagation.The lacObindingsitefortheLacI
repressorwouldallowforaffinitypurificationof theplasmidswithSNAP-taggedLacIbecause
the SNAP tag allows for covalent attachment of a biotin-fluor tag called BG-biotin-649. In
conjugating this tag to yeast extracts, LacI would be functionalized with both biotin for
attachment to a flow-cell surface and a red fluor for visualization. This systemwould purify
singlemoleculesandtetherthemtoaflow-cell;GFPlabeledMCMscouldthenbecountedby
wayofquantitativephotobleachingusingTIRFmicroscopy.
Originally,theprojectwasbasedoffofthestrainbuiltfromyFS961,butthepurification
system failed and MCMs could not be quantified for several reasons. Not only was the
purification system not yet optimized for the complex, but also unreacted dye could not be
removedfromtheyeastextractandpurificationwascomplicated.
ItwasatthispointintheprocessthatyFS833wasusedinconjunctionwithpFS270to
makeyFS989andpFS458andpFS466wereusedtocreatetheLacI-SNAPplasmidthatwewere
able tomoveforwardwith. InchangingtheMCM4-GFPtagging,we inserteda10aminoacid
linkerbetweenthegenesinordertoincreasethefunctionalityoftheGFPtag.Increatinganew
LacI-SNAP plasmid, we were able to confirm that these genes were within reading frame,
allowingfortheLacI-SNAPfusionproteintobetranslatedcorrectlyonceinsertedintotheyeast
genome.
For the future directions of this project, the TALO8 plasmidwill be transformed into
yFS990 and this strain will be used to optimize extract purification. Once the purification is
successful and singleplasmidmolecules canbe immobilized,TIRFmicroscopywillbeused to
quantifythenumberofGFPfluorescentMCM4sboundtoARS1bywayofphotobleaching.With
single-moleculequantitationoptimized,notonlywill newyeast strainswill beproduced that
22
containmutationstotheB1andB2elementswithinknownoriginsequences,butalsoknown
later-firingARSoriginswillbeimplementedinthesysteminordertodeterminetheregulation
ofMCMloading.
Determining the number of MCMs bound to specific origins, both endogenous and
mutated, will help test the hypothesis that the number of MCMs loaded on origins of
replicationdeterminesfiringefficiencyandtiming.Thisprocessissignificantbecausethetiming
of genome replication in S phase has been correlated with gene expression, cellular
differentiation, development, and DNA repair. Orderly replication is necessary to not only
maintaingenomestability,butalsoregulatetheeventsofgenomemetabolismtoconductthe
chemicalprocessesthatmaintainalllivingcells.
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