Assay for Detection of Homologous DNA Interactions

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Project Summary Homologous DNA interactions are a key determinant of chromosome structure and genome function. In mitotic cells, pairing between sister chromatids ensures faithful chromosome segregation and efficient chromosome break repair. In the germ line, pairing between homologous chromosomes is a precondition for genetic exchange during meiosis, ensuring segregation of homologous chromosomes and creation of novel allele combinations. Defects in homologous DNA pairing contribute to chromosome missegregation and gross chromosome rearrangements, conditions associated with cancer, premature aging and birth defects. Moreover, allele choice for monoallelic gene expression is also thought to involve transient interactions between homologous DNA segments. Our long-term goal is to understand the molecular mechanism of homologous pairing and its role in chromosome structure and function. We hypothesize that pairing preferentially occurs in genetically determined chromosome regions separated by loops where pairing is low or absent. Our investigation focuses on the development of a genome-wide assay for detection of homologous DNA interactions in budding yeast as a model organism. We have developed the Homologous Pairing Capture (HPC) assay system that allows identification of homologous DNA interactions in intact cells, on a genome-wide scale and at nucleotide resolution. In our Specific Aim 1, we will establish a prototype for detecting and quantitating DNA pairing interactions along maximally paired yeast chromosomes during meiosis. We will further map preferred associations between homologous chromosomes. In our Specific Aim 2, we will develop approaches to quantitatively distinguish pairing interactions between similar from those between identical DNA segments. This will enable us to distinguish pairing interactions between homologous chromosomes from those occurring between sister chromatids. To provide independent assay validation, we will apply HPC to branched recombination intermediates that should correlate with known positions of genetic exchange. Identification of preferred pairing sequences via the HPC assay will lay the foundation for a mechanistic understanding of this ubiquitous biological process.