Analysis of Cohesin Association to Newly Replicated DNA Through Nascent Strand Binding Assay (NSBA).

Abstract

Replication forks engage chromatin-bound cohesin complexes during chromosome replication. Interfacing between cohesin and replication forks influences both cohesion establishment and fork functionality. However, the mechanisms mediating this process are scarcely understood. Here we describe the nascent strand binding assay (NSBA) methodology, developed in budding yeast to discriminate the association of cohesin to either parental unreplicated or nascent DNA in the environment of replication forks. NSBA quantitatively estimates the association of a protein of interest to newly replicated DNA. For this, nascent strands are in vivo labeled with the thymine analogue bromodeoxyuridine and chromatin is immunoprecipitated to isolate a fraction enriched in DNA associated to the target protein. The abundance of nascent DNA is then assessed through BrdU immunoprecipitation followed by quantitative PCR, allowing for the parallel analysis of diverse genomic regions. While originally employed to characterize the association of cohesin to nascent sister chromatids, NSBA can be applied to study other factors dynamically associating to nascent DNA.