The goal of the proposed research is to develop and apply a new quantitative method of assaying DNA repair efficiency in vivo, suitable for diagnostic use in readily available patient material. Genomic stability of living cells is continuously threatened from without, by environmental genotoxins as well as from within, by reactive metabolites and enzymatic malfunction. Cells have evolved multiple mechanisms to repair a broad spectrum of damages to DNA, and it is generally thought that small genetic or epigenetic variations in the proficiency of DNA repair may have a profound lifetime impact on genomic stability of an organism. Recent years have seen an exponential increase in studies that connect DNA repair gene single nucleotide polymorphisms (SNPs), expression levels, or methylation status to cancer risk or therapeutic outcome. Understanding the functional, mechanistic significance of the genotypic, epigenetic, or transcriptome signatures found associated with disease risk should deepen our knowledge of the disease process, validate association studies and, ultimately, inform future clinical decisions. In the past several years I have adopted a high resolution, global DNA analysis tool - microfluidics-assisted display of stretched DNA molecules - and used it for a quantitative analysis of DNA replication in vivo and its response to DNA damage in human cells. In this proposal I aim to adapt this technology to a new, thus far unrealized application -- to measure DNA repair in vivo. I will develop, validate, and apply an adaptation of our DNA-stretching technology to study DNA repair using base excision repair (BER) of methyl adducts in mouse primary fibroblasts as a model system. Using cells with dosage or mutation defects in XRCC1, a protein critical for BER, I will determine whether my approach allows measuring relatively small variations in BER efficiency. At the next stage, I will test whether I can apply my technology to measure DNA repair in clinically relevant samples such as human peripheral blood mononuclear cells and human lymphoblastoid cell lines homozygous for the SNPs in XRCC1 that are associated with increased risk of cancer. Finally, I will take steps to determine applicability of my technology to the study of DNA repair systems other than BER.
The goal of the proposed research is to develop and apply a new quantitative and sensitive method of assaying DNA repair efficiency in living cells, suitable for diagnostic use in samples that can be made available from patients. We believe this assay will contribute to the understanding of the functional significance of the small genetic or epigenetic changes in DNA repair genes that are found associated with an increased risk of cancer, and thus validate associations found in epidemiological studies and inform clinical decisions. In addition, this assay may be adapted to use with clinical specimens of tumors to help devise individualized chemotherapeutic strategies.