A key event in eukaryotic DNA replication is origin licensing in G1-phase, during which two Mcm2-7 replicative DNA helicases are loaded onto each origin DNA in an inactive, head-to-head fashion. Origin licensing marks every potential origin in a cell, and the opposing orientation of the loaded helicases ensures that they are poised to initiate bidirectional replication when the cell enters S-phase. Although it has long been known that the origin-recognition complex (ORC) binds origin DNA to direct helicase loading, the molecular mechanism by which two oppositely oriented helicases are loaded remains puzzling. Previous biochemical studies found evidence in support of a two-ORC mechanism for helicase loading wherein each of the two Mcm2-7 helicases are recruited by a separate, oppositely oriented ORC molecule. In contrast, single-molecule and cryo-EM approaches observed predominantly one ORC involved in helicase loading, but could not explain how a single ORC could load two oppositely oriented helicases.
In this paper, a collaboration with Steve Bell’s lab at MIT, Ph.D. student Shalini Gupta reconciles these seemingly contradictory observations. Using single-molecule fluorescence energy transfer (sm-FRET), she observed interactions in vitro between individual ORC molecules and the Mcm2-7 helicases in real time at two separate interfaces. In the large majority of instances, a single ORC molecule recruits both Mcm2-7 helicases through direct interactions. Between recruitment of the first and the second helicase, ORC ‘flips’ its orientation on DNA using a flexible protein tether to the first loaded Mcm2-7. This remarkable ORC inversion ensures that the two helicases are recruited via similar interactions, but in opposite orientations. The data define a complete, integrated pathway for helicase loading that resolves the apparent contradictions between previous observations. The tethered-flip mechanism provides a molecular explanation for how cells avoid the potentially damaging consequences of incompletely-formed helicase pairs at origins.
Gupta S., et al. A helicase-tethered ORC flip enables bidirectional helicase loading
eLife 10, e74282 (2021)
This article was the subject of an eLife “Insight article” by Bruce Stillman.
Replication of chromosomal DNA in eukaryotes has two major stages. Starting in the G1 phase of the cell cycle, double hexamers consisting of two copies of the Mcm2-7 replicative helicase are assembled at replication origins. Later, in S phase, the two helicases are incorporated into two oppositely oriented CMG (Cdc45-Mcm2-7-GINS) complexes that each then form the core of a replisome. Control of this “activation” step, which is triggered by the protein kinases DDK and S-CDK, is essential to ensure that each part of the genome is replicated once and only once in each cell cycle.
In this paper, Steve Bell’s and Jeff Gelles’ labs used multi-wavelength single-molecule fluorescence colocation (“CoSMoS”) methods to study in vitro the molecular mechanism of the activation process. The journal’s acceptance summary notes that “The manuscript provides new and convincing evidence that a heretofore unknown intermediate state [called “CtG”] for replication start contains multiple copies of the GINS and Cdc45 proteins prior to initiation at each origin with one double hexamer of the MCM2-7 complex. The number of GINS and Cdc45 is determined by DDK phosphorylation of the MCM’s and the probability to create an active helicase (CMG) is increased with multiple numbers of the bound ancillary factors…. The single molecule studies and biochemistry are beautifully executed providing the evidence for such intermediates…. The addition of in vivo studies demonstrates that modulating the multiplicity of DDK phosphorylation (and proposed, CtG formation) has an impact on origin usage in cells.”
together with collaborators from 10.7554/eLife.65471
Kim L.D.J., et al., DDK regulates replication initiation by controlling the multiplicity of Cdc45-GINS binding to Mcm2-7.
eLife 10, e65471 (2021)
In this project, Kankowan Champasa from Stephen Bell’s lab at MIT collaborated with other researchers from the Bell and Gelles labs to study a key process that sets the stage for replication of chromosomal DNA. They explain “licensing of eukaryotic origins of replication requires DNA loading of two copies of the Mcm2-7 replicative helicase to form a head-to-head double-hexamer, ensuring activated helicases depart the origin bidirectionally.” The researchers identified a conserved motif in the Mcm4 helicase subunit essential for formation of productive replication complexes. Single-molecule fluorescence energy transfer experiments show that mutations in the motif still allow the two hexamers to come into contact, but they prevent the formation of the stable double-hexamers that perform the extensive DNA unwinding needed for replication.
A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding.
Champasa, K., Blank, C., Friedman, L.J., Gelles, J., and Bell, S.P.
eLife (2019) 8:e40576
Grp94 is a molecular chaperone that helps to fold and maintain the folded state of “client” proteins in the endoplasmic reticulum. Acceleration of client folding is driven by conformational changes in Grp94. However, the sequence of conformational changes and how these changes are coupled to the cycle of ATP hydrolysis is not well understood. Prof. Timothy Street and his lab members Bin Huang and Ming Sun, in collaboration with Larry Friedman, did single-molecule fluorescence resonance energy transfer (FRET) experiments to directly observe conformational cycling in individual Grp94 molecules. Their studies show that ATP hydrolysis can drive repeated cycling between alternative “closed” states of Grp94, suggesting a way that enzyme might propagate structural changes to client molecules.
Conformational Cycling within the Closed State of Grp94, an Hsp90-Family Chaperone
Huang, B., Friedman, L.J., Gelles, J., Sun, M., and Street, T.O.
Journal of Molecular Biology 431, 3312-3323 (2019).