LAUSR

Protein–DNA interactions are dynamic, and these dynamics are an important aspect of chromatin‐associated processes such as transcription or replication. Due to a lack of methods to study on‐ and off‐rates across entire genomes, protein–DNA interaction dynamics have not been studied extensively. Here, we determine in vivo off‐rates for the Saccharomyces cerevisiae chromatin organizing factor Abf1, at 191 sites simultaneously across the yeast genome. Average Abf1 residence times span a wide range, varying between 4.2 and 33 min. Sites with different off‐rates are associated with different functional characteristics. This includes their transcriptional dependency on Abf1, nucleosome positioning and the size of the nucleosome‐free region, as well as the ability to roadblock RNA polymerase II for termination. The results show how off‐rates contribute to transcription factor function and that DIVORSEQ (Determining In Vivo Off‐Rates by SEQuencing) is a meaningful way of investigating protein–DNA binding dynamics genome‐wide.