DNSN-1 recruits GINS for CMG helicase assembly during DNA replication initiation in <i>Caenorhabditis elegans</i>

Assembly of the CMG (CDC-45-MCM-2-7-GINS) helicase is the key regulated step during eukaryotic DNA replication initiation. Until now, it was unclear whether metazoa require additional factors that are not present in yeast. In this work, we show that Caenorhabditis elegans DNSN-1, the ortholog of human DONSON, functions during helicase assembly in a complex with MUS-101/TOPBP1. DNSN-1 is required to recruit the GINS complex to chromatin, and a cryo-electron microscopy structure indicates that DNSN-1 positions GINS on the MCM-2-7 helicase motor (comprising the six MCM-2 to MCM-7 proteins), by direct binding of DNSN-1 to GINS and MCM-3, using interfaces that we show are important for initiation and essential for viability. These findings identify DNSN-1 as a missing link in our understanding of DNA replication initiation, suggesting that initiation defects underlie the human disease syndrome that results from DONSON mutations.</p

Structural Basis for Inhibition of Human Primase by Arabinofuranosyl Nucleoside Analogues Fludarabine and Vidarabine.

Nucleoside analogues are widely used in clinical practice as chemotherapy drugs. Arabinose nucleoside derivatives such as fludarabine are effective in the treatment of patients with acute and chronic leukemias and non-Hodgkin's lymphomas. Although nucleoside analogues are generally known to function by inhibiting DNA synthesis in rapidly proliferating cells, the identity of their in vivo targets and mechanism of action are often not known in molecular detail. Here we provide a structural basis for arabinose nucleotide-mediated inhibition of human primase, the DNA-dependent RNA polymerase responsible for initiation of DNA synthesis in DNA replication. Our data suggest ways in which the chemical structure of fludarabine could be modified to improve its specificity and affinity toward primase, possibly leading to less toxic and more effective therapeutic agents.Boehringer-Ingelheim Fonds PhD Fellowship Janggen-Pöhn- Stiftung Award Swiss National Science Foundation Award (all to Sandro Holzer

A conserved mechanism for regulating replisome disassembly in eukaryotes.

Replisome disassembly is the final step of eukaryotic DNA replication and is triggered by ubiquitylation of the CDC45-MCM-GINS (CMG) replicative helicase1-3. Despite being driven by evolutionarily diverse E3 ubiquitin ligases in different eukaryotes (SCFDia2 in budding yeast1, CUL2LRR1 in metazoa4-7), replisome disassembly is governed by a common regulatory principle, in which ubiquitylation of CMG is suppressed before replication termination, to prevent replication fork collapse. Recent evidence suggests that this suppression is mediated by replication fork DNA8-10. However, it is unknown how SCFDia2 and CUL2LRR1 discriminate terminated from elongating replisomes, to selectively ubiquitylate CMG only after termination. Here we used cryo-electron microscopy to solve high-resolution structures of budding yeast and human replisome-E3 ligase assemblies. Our structures show that the leucine-rich repeat domains of Dia2 and LRR1 are structurally distinct, but bind to a common site on CMG, including the MCM3 and MCM5 zinc-finger domains. The LRR-MCM interaction is essential for replisome disassembly and, crucially, is occluded by the excluded DNA strand at replication forks, establishing the structural basis for the suppression of CMG ubiquitylation before termination. Our results elucidate a conserved mechanism for the regulation of replisome disassembly in eukaryotes, and reveal a previously unanticipated role for DNA in preserving replisome integrity

Keeping It Together and Taking It Apart: Structural Investigations of Replisome Complexes Involved in DNA Replication Fork Protection and Termination

DNA replication is an essential cellular process whose dysregulation is implicated in severe human disease, including cancer. Broadly, DNA replication involves the unwinding of the DNA double-helix, allowing DNA polymerases to use each unwound strand as a template for nascent DNA synthesis; the complex molecular machinery responsible for DNA replication is known as the replisome. However, during unwinding and DNA synthesis, the replisome may encounter various obstacles such as DNA damage and tightly-bound proteins, necessitating specific pathways tailored to tolerating and overcoming such hinderances. Upon completion of DNA replication, the replisome is disassembled in a regulated manner. An understanding of DNA replication requires structural knowledge of how the various replisome components assemble to form the replicative machinery. Although significant advances have been made in recent years, facilitated by the development of electron cryo-microscopy (cryo-EM), our understanding of replisome structure remains in its infancy. Here I present high-resolution cryo-EM structures of the most complete replisome complexes to date, which are involved in multiple aspects of DNA replication and replication-coupled processes. First, I describe how four replisome components – the fork protection complex (Csm3-Tof1-Mrc1) plus Ctf4 – associate with the replicative helicase CMG. The fork protection complex is involved in achieving maximal rates of DNA replication, as well as performing roles in protecting replication forks from varied forms of replication stress. Ctf4 acts as a structural hub recruiting factors required for cohesion establishment and epigenetic inheritance. Second, I discuss structural insights into the regulation of replisome disassembly as the final stage of DNA replication, presenting the first structure of a replisome complex which has translocated onto double-stranded DNA and is bound by the termination-specific E3 ubiquitin ligase, SCFDia2