Assay for Detection of Homologous DNA Interactions

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.

Grant Number: 5R01GM141698-04
NIH Institute/Center: NIH
Principal Investigator: Valentin Boerner