A conserved mechanism for regulating replisome disassembly revealed by cryo-EM - Tom Deegan

Michael Jenkyn-Bedford1, 4, Morgan L. Jones1, 4, Yasemin Baris1, Karim P.M. Labib2, Giuseppe Cannone1, Joseph T.P. Yeeles1, 5 & Tom D. Deegan3, 5, * 1 MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH 2 MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, School of Life Sciences, University of Dundee, Dundee, DD1 5EH 3 MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Western General Hospital, Crewe Road South, Edinburgh, EH4 2XU 4 Equal contribution 5 Co-corresponding Presenting Eukaryotic DNA replication is driven by a highly complex and dynamic macromolecular machine called the replisome. The replisome is assembled at distinct genomic loci termed DNA replication origins during replication initiation, and then remains stably associated with replication forks, until two forks emanating from adjacent replication origins converge, or a single fork reaches the end of a linear chromosome, at which point replication terminates. Upon termination, the replisome is disassembled in two steps: first, the CMG (Cdc45-MCM-GINS) replicative helicase, which forms the core of the replisome, is ubiquitylated by a cullin-RING type E3 ubiquitin ligase (SCFDia2 in yeasts, CUL2Lrr1 in metazoa); then, the ubiquitylated CMG-replisome is disassembled by the Cdc48 / p97 ATPase. As there is no known mechanism for origin independent replisome assembly in S-phase, premature replisome disassembly must be avoided, to prevent replication fork collapse and genome instability. Biochemical evidence has suggested that the ‘excluded’ DNA strand, which forms the template for lagging strand synthesis at all replication forks and is lost upon termination, inhibits CMG ubiquitylation during elongation. However, the molecular basis for how SCFDia2 and CUL2Lrr1 discriminate terminated from elongating replisomes, and selectively ubiquitylate CMG only after termination, has remained unknown, largely due to a lack of structural information. We have solved high resolution cryo-EM structures of both yeast and human replisome-E3 ubiquitin ligase assemblies, representing bona fide termination intermediates. Our structures, together with functional in vitro and in vivo analyses, identify a novel regulated interaction between E3 ligase and CMG, which is essential for CMG ubiquitylation and, in turn, replisome disassembly. This interface underpins a simple but previously undescribed molecular mechanism for the regulation of replisome disassembly, which is conserved between yeast and metazoa.