ABSTRACT DNA repair is a crucial mechanism for maintaining genomic stability in cells. Defects in the DNA repair machinery increase cell vulnerability to DNA-damaging agents and accumulation of mutations in the genome, and lead to the development of various disorders including cancers. Studies that have measured DNA repair capacity (DRC), including our own, have estimated a much higher risk of breast cancer (BC) (3-15-fold) than most other established risk factors for BC, with the exception of highly penetrant mutations in genes like BRCA1 and BRCA2, genes critical to DNA repair. Despite the strength of this association, no large-scale prospective studies of BC exist. Even though some BC risk models include known mutations in DNA repair genes, genotype only partially explains phenotype, and BC risk models currently do not include phenotypic DNA repair measures. The lack of inclusion of a major risk factor – DRC – is likely the major reason that clinical BC risk models have only modest performance - which makes it very challenging to target effective primary prevention options (e.g., chemoprevention) for the majority of women who are not known mutation carriers. Further, secondary prevention options (e.g., onset, frequency, and method of BC screening by mammography or other supplemental methods) could be targeted more efficiently if more accurate risk assessment existed. The main limitation of use of DRC for targeted prevention has been the lack of a high-throughput DRC assay, in particular a phenotypic DRC assay, for integration into cancer risk assessment. We have overcome this major gap by adapting our high-throughput, fully-automated ɣ-H2AX assay system which was originally designed for assaying DNA double strand breaks (DSB) in freshly-drawn blood for use with archival blood samples. We propose one of the largest prospective studies estimating the effect of DSB repair using an enriched cohort (n=12,563) that spans the spectrum of absolute BC risk. Using a nested case-control design within this cohort (699 cases, 1:1 match), we will measure DSB-DRC in archival biospecimens collected at baseline (Aim 1a). We will optimize the assay protocol for measuring DSB-DRC using fresh fingerstick blood and measure longitudinal changes in DSB-DRC in young women (age <40 years) (Aim 1b) (n=100, 1-2 years apart). We will then comprehensively assess the independent contribution of DSB-DRC over genetic and epigenetic alterations in DSB repair genes, and assess and whether genetic and epigenetic changes interact with DSB-DRC in increasing BC risk (Aim 2). We will investigate the clinical utility of DSB-DRC by quantifying the improvement in standard BC risk model performance from its inclusion (Aim 3a), and evaluating the association between DSB-DRC and 5 year survival after BC diagnosis (Aim 3b). Our study will provide essential empirical evidence from integrating functional assays into population studies to accelerate targeted prevention options linked to aber...