Abstract
In eukaryotes, DNA replication requires the origin recognition complex (ORC), a six-subunit assembly that promotes replisome formation on chromosomal origins. Despite extant homology between certain subunits, the degree of structural and organizational overlap between budding yeast and metazoan ORC has been unclear. Using 3D electron microscopy, we determined the subunit organization of metazoan ORC, revealing that it adopts a global architecture very similar to the budding yeast complex. Bioinformatic analysis extends this conservation to Orc6, a subunit of somewhat enigmatic function. Unexpectedly, a mutation in the Orc6 C-terminus linked to Meier-Gorlin syndrome, a dwarfism disorder, impedes proper recruitment of Orc6 into ORC; biochemical studies reveal that this region of Orc6 associates with a previously uncharacterized domain of Orc3 and is required for ORC function and MCM2-7 loading in vivo. Together, our results suggest that Meier-Gorlin syndrome mutations in Orc6 impair the formation of ORC hexamers, interfering with appropriate ORC functions. DOI:http://dx.doi.org/10.7554/eLife.00882.001.
Highlights
The faithful inheritance of genetic information depends on the precise replication of DNA; errors such as under- or over-replication of DNA can lead to genetic instabilities, malignant transformation, or cell death (Klingseisen and Jackson, 2011; Abbas et al, 2013)
Orc6 transgenes containing mutations in the conserved C-terminal tail fail to incorporate into DmORC in vivo, and do not complement an orc6-null allele when introduced into flies. These results suggest that the MGS mutation in Orc6 directly affects the integrity of origin recognition complex (ORC), and indicate that destabilization of ORC contributes to the pathogenesis of Meier-Gorlin syndrome caused by mutations in Orc6, interfering with pre-replicative complex (pre-RC) assembly and origin licensing
A majority of the components of the core DNA replication initiation machinery appear evolutionary conserved across eukaryotes, suggesting that structural and mechanistic aspects of replicative helicase loading are as well
Summary
The faithful inheritance of genetic information depends on the precise replication of DNA; errors such as under- or over-replication of DNA can lead to genetic instabilities, malignant transformation, or cell death (Klingseisen and Jackson, 2011; Abbas et al, 2013). Replication initiation machineries are loaded onto DNA during the G1-phase of the cell cycle, ‘licensing’ origins of replication for subsequent replisome assembly in S-phase (Masai et al, 2010; Abbas et al, 2013). The MCM complex is loaded onto double-stranded DNA as an inactive double-hexamer (Evrin et al, 2009; Remus et al, 2009; Gambus et al, 2011), and is activated only after origin firing in the subsequent S-phase to commence DNA replication (Vijayraghavan and Schwacha, 2012).
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