DONSON is required for CMG helicase assembly in the mammalian cell cycle

Abstract

DONSON is one of 13 genes mutated in a form of primordial microcephalic dwarfism known as Meier‐Gorlin syndrome. The other 12 encode components of the CDC45‐MCM‐GINS helicase, around which the eukaryotic replisome forms, or are factors required for helicase assembly during DNA replication initiation. A role for DONSON in CDC45‐MCM‐GINS assembly was unanticipated, since DNA replication initiation can be reconstituted in vitro with purified proteins from budding yeast, which lacks DONSON. Using mouse embryonic stem cells as a model for the mammalian helicase, we show that DONSON binds directly but transiently to CDC45‐MCM‐GINS during S‐phase and is essential for chromosome duplication. Rapid depletion of DONSON leads to the disappearance of the CDC45‐MCM‐GINS helicase from S‐phase cells and our data indicate that DONSON is dispensable for loading of the MCM2‐7 helicase core onto chromatin during G1‐phase, but instead is essential for CDC45‐MCM‐GINS assembly during S‐phase. These data identify DONSON as a missing link in our understanding of mammalian chromosome duplication and provide a molecular explanation for why mutations in human DONSON are associated with Meier‐Gorlin syndrome.

Starting DNA Synthesis: Initiation Processes during the Replication of Chromosomal DNA in Humans

“…These studies, in combination with cell biological studies and advanced modern technologies, including next generation sequencing, as well as structural biology techniques, have yielded new insights into human DNA replication [ 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 ]. Although origin-dependent replication initiation processes in higher eukaryotes are not as well understood as in yeast, combining the results of multiple model systems has yielded a picture of highly conserved processes in combination with subtle changes as discussed below (summarised in Figure 1 , [ 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 24 , 25 , 26 , 27 , 28 ]).…”

“…Then, TOPBP1 binds to the GINS complex and P-Treslin bringing the GINS proteins to the MCM-DHs located at origin sequences. This complex formation allows CDC45 to associate with GINS and MCM2-7 forming the CMG complex, the eukaryotic replicative helicase [ 22 , 24 , 25 , 26 , 27 , 28 ]. Next, TOPBP1 and the Treslin–MTBP complex leave the origin-bound CMG complexes, but two Donson molecules remain associated with each active CMG complex [ 22 , 28 ].…”

“…DNA replication is a highly conserved process at molecular levels in all eukaryotes [ 1 , 2 , 3 , 4 , 5 , 6 , 8 , 9 ]. However, differences among human, yeast, and other eukaryotes exist and have been studied [ 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ]. For example, origins of replication have specific sequence requirements in some yeast, such as Saccharomyces cerevisiae , whereas in most other eukaryotes, replication origins are defined by protein–DNA complexes with little or no dependence on the DNA sequence [ 15 ].…”

“…In yeast, DNA polymerase ε (Pol ε) and MCM10 (minichromosome maintenance 10) support its assembly and activation, whereas in multicellular organisms, the protein Donson, a protein not yet found in S. cerevisiae , assists in the assembly of the CMG complex [ 28 ]. Interestingly, mutations in ORC subunits, CDC6 (cell division cycle 6), CDT1 (chromatin licensing and DNA replication factor 1), Donson, MCM2-7, CDC45, and other replication initiation factors have been found to be the cause of genetic diseases, such as Meier–Gorlin syndrome, highlighting the importance of understanding the mechanism of initiation in humans [ 9 , 22 , 24 , 25 , 26 , 27 , 28 ].…”

Combination of AID2 and BromoTag expands the utility of degron-based protein knockdowns

“…A protein fused with BromoTag is recruited to CRL2–VHL in the presence of AGB1 for rapid proteolysis via the UPS. Both dTAG and BromoTag have been used in many studies, indicating that they are also promising conditional degrons (Evrin et al , 2023; Mylonas et al , 2021; Olsen et al , 2022; Weintraub et al , 2017). However, few studies have compared conditional degrons to discern their similarities and differences, and BromoTag has not yet been included for comparison (Bondeson et al , 2022; Noviello et al , 2023).…”