Single-copy locus proteomics of early- and late- firing DNA replication origins identifies a role of Ask1/DASH complex in replication timing control

The chromatin environment at origins of replication is thought to influence DNA replication initiation in eu-karyotic genomes. However, it remains unclear how and which chromatin features control the firing of early-efficient (EE) or late-inefficient (LI) origins. Here, we use site-specific recombination and single-locus chromatin isolation to purify EE and LI replication origins in Saccharomyces cerevisiae. Using mass spec-trometry, we define the protein composition of native chromatin regions surrounding the EE and LI replication start sites. In addition to known origin interactors, we find the microtubule-binding Ask1/DASH complex as an origin-regulating factor. Strikingly, tethering of Ask1 to individual origin sites advances replication timing (RT) of the targeted chromosomal domain. Targeted degradation of Ask1 globally changes RT of a subset of or-igins, which can be reproduced by inhibiting microtubule dynamics. Thus, our findings mechanistically con-nect RT and chromosomal organization via Ask1/DASH with the microtubule cytoskeleton.