RESEARCHARTICLES

Cell-wideanalysisofproteinthermalunfoldingrevealsdeterminantsofthermostability

Temperature-inducedcelldeathisthoughttobeduetoproteindenaturation,butthedeterminantsofthermalsensitivityofproteomesremainlargelyuncharacterized.Wedevelopedastructuralproteomicstrategytomeasureproteinthermostabilityonaproteome-widescaleandwithdomain-levelresolution.WeappliedittoEscherichiacoli,Saccharomycescerevisiae,Thermusthermophilus,andhumancells,yieldingthermostabilitydataformorethanproteins.Ourresults(i)indicatethattemperature-inducedcellularcollapseisduetothelossofasubsetofproteinswithkeyfunctions,(ii)shedlightontheevolutionaryconservationofproteinanddomainstability,and(iii)suggestthatnativelydisorderedproteinsinacellarelessprevalentthanpredictedand(iv)thathighlyexpressedproteinsarestablebecausetheyaredesignedtotoleratetranslationalerrorsthatwouldleadtotheaccumulationoftoxicmisfoldedspecies.

The[4Fe4S]clusterofhumanDNAprimasefunctionsasaredoxswitchusingDNAchargetransport

DNAchargetransportchemistryoffersameansoflong-range,rapidredoxsignaling.Wedemonstratethatthe[4Fe4S]clusterinhumanDNAprimasecanmakeuseofthischemistrytocoordinatethefirststepsofDNAsynthesis.UsingDNAelectrochemistry,wefoundthatachangeinoxidationstateofthe[4Fe4S]clusteractsasaswitchforDNAbinding.Single-atommutationsthatinhibitthischargetransferhinderprimaseinitiationwithoutaffectingprimasestructureorpolymerization.Generatingasinglebasemismatchinthegrowingprimerduplex,whichattenuatesDNAchargetransport,inhibitsprimertruncation.Thus,redoxsignalingby[4Fe4S]clustersusingDNAchargetransportregulatesprimasebindingtoDNAandillustrateschemistrythatmayefficientlydrivesubstratehandoffbetweenpolymerasesduringDNAreplication.

REVIEW

MechanismsforinitiatingcellularDNAreplication

CellularDNAreplicationfactoriesdependonring-shapedhexamerichelicasestoaidDNAsynthesisbyprocessivelyunzippingtheparentalDNAhelix.ReplicativehelicasesareloadedontoDNAbydedicatedinitiator,loader,andaccessoryproteinsduringtheinitiationofDNAreplicationinatightlyregulated,multistepprocess.WediscussherethemolecularchoreographyofDNAreplicationinitiationacrossthethreedomainsoflife,highlightingsimilaritiesanddifferencesinthestrategiesusedtodepositreplicativehelicasesontoDNAandtomelttheDNAhelixinpreparationforreplisomeassembly.Althoughinitiatorsandloadersarephylogeneticallyrelated,themechanismstheyuseforac







































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