Summary:TheobjectiveofthisproposalistounderstandthemechanismsthatgovernDNAreplicationfork stability upon treatment with multiple-drug doses in BRCA-mutant tumors. Mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 are associated with several forms of cancer, including breast and ovariancancers.BRCAproteinsarerequiredforthemaintenanceofreplicationforkstabilityfollowingtreatment withchemotherapeuticssuchascisplatin,aDNAcross-likingagentwidelyusedforcancertreatment.Replication forkscanreversetoaidtherepairofDNAdamageinducedbychemotherapeuticsandBRCAproteinsarekey to protecting the reversed structures from nucleolytic degradation. In absence of BRCA, reversed replication forksareextensivelydegradedbynucleases,leadingtochemosensitivity.However,themolecularbasisofthe DNA-damaging drug sensitivity inBRCA-mutant tumors remainunclear. Defining these mechanisms is crucial forbasicresearchtoinformandimprovecurrentclinicaloncologyregimensbasedonDNAreplicationinhibitors. Most studies focus on the analysis of replication perturbations following a single-dose treatment. For the first time,weinvestigatedreplicationforkperturbationsinBRCA1-deficientcellstreatedwithcisplatin24hoursafter pre-exposure to this drug. Our preliminary data challenge the dogma that DNA-damaging drug sensitivity originatesfromtheextendedreplicationforkdegradationphenotypeobservedafterasingle-dosetreatmentin BRCA1-deficient cells. We found that fork degradation is no longer detectable when using multiple cisplatin doses.ThiseffectdependsontheoverexpressionandDNAprimaseactivityofthePrimPolpolymerase.Based on this premise, we hypothesize that a PrimPol-dependent pathway rescues replication fork degradation followingmultipleroundsofcisplatintreatmentandmodulatescisplatinsensitivityinBRCA1-deficientcells.We alsopositthatcancercellrelianceonforkreprimingisenhancedunderanyconditionthatleadstoreversedfork degradation?e.g.,BRCA1orBRCA2proteindeficiency. Aim 1 will define the function of the dual enzymatic activity of PrimPol in replication fork stability in BRCA1- deficientcellsfollowingtreatmentwithmultiplecisplatindoses.Aim2willdeterminewhetherPrimPol-mediated repriming rescues fork degradation by suppressing fork reversal, which would otherwise lead to extensive nascentstranddegradationinBRCA-mutants.Aim3willdeterminetheimpactofthecisplatin-inducedPrimPol overexpressionongenomicinstabilityandBRCA1-deficentcancercellviability.Thiswillbeachievedbyusinga unique combination of single-molecule DNA replication and electron microscopy approaches available in our laboratory.ThesestudieswillestablishanewparadigmforthePrimPolpolymeraseinreplicationforkprotection and genomic stability. They will also introduce the novel concept that multiple-drug doses need to be used to fully understand how cells respond to chemotherapeutics, and they will offer new insights for the treatment of BRCA-mutantcancerpatientsbytargetingthePrimPol-dependentpathway.