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

16

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

17

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.

20

Figure8.Plasmidconstructionprocessandconfirmation Figure9.Summaryoftransformation

TheLacI-SNAPplasmidwasthentransformedintoyPF989tocreatethestrainyFS990.

PCRtaggingofpFS458forLacIandothergenes

PCRtaggingofpFS466forSNAP

GibsonassemblyofPCRproductsandbacterial

transformationintoE.coli

SelectforAmp+coloniesbygrowingcandidatesinLBbroth

withampicillin

Alkalinelysisminipreptoobtainplasmid

SendplasmidoutforsequencinganddigestplasmidwithEcoRVtoconPirmplasmidsequence

DigestplasmidwithBsm1tolinearizeforyeasttransformation

Uncut,EcoRV,Bsm1

21

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.

23

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