RT Journal Article SR Electronic T1 Drosophila SUMM4 complex couples insulator function and DNA replication timing control JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.10.02.462895 DO 10.1101/2021.10.02.462895 A1 Evgeniya N. Andreyeva A1 Alexander V. Emelyanov A1 Markus Nevil A1 Lu Sun A1 Elena Vershilova A1 Christina A. Hill A1 Michael-C. Keogh A1 Robert J. Duronio A1 Arthur I. Skoultchi A1 Dmitry V. Fyodorov YR 2022 UL http://biorxiv.org/content/early/2022/07/11/2021.10.02.462895.abstract AB Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early versus late in different cell types remain incompletely understood. Drosophila SNF2-related factor SUUR imparts under- replication of late-replicating intercalary heterochromatin in polytene chromosomes. SUUR negatively regulates DNA replication fork progression; however, its mechanism of action remains obscure. Here we developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates SUUR ATPase activity and is required for a normal spatiotemporal distribution of SUUR in vivo. SUUR and Mod(Mdg4)-67.2 together mediate the activities of gypsy insulator that prevent certain enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, SuUR or mod(mdg4) mutations reverse under-replication of intercalary heterochromatin. Thus, SUMM4 can impart late replication of intercalary heterochromatin by attenuating the progression of replication forks through euchromatin/heterochromatin boundaries. Our findings reveal that DNA replication can be delayed by a chromatin barrier and uncover a critical role for architectural proteins in replication control. They suggest a mechanism for replication timing that does not depend on an asynchronous firing of replication origins.Competing Interest StatementCompeting interests LS and MCK are employed by Epicypher, Inc., a commercial developer and supplier of the EpiDyne nucleosomes and associated remodeling assay platforms used in this study. The remaining authors declare no competing interests.