Tag Archives: yeast

A helicase-tethered ORC flip enables bidirectional helicase loading

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 eLifeInsight article” by Bruce Stillman.

Single-molecule studies reveal branched pathways for activator-dependent assembly of RNA polymerase II pre-initiation complexes

A key event leading to the synthesis of a eukaryotic messenger RNA  is the assembly of a pre-initiation complex (PIC) on promoter DNA near the transcription start site.  The PIC contains RNA polymerase II (pol II) plus general transcription factors TFIID, TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH.  PIC assembly is enhanced by binding of transcription activator proteins to separate DNA sites called upstream activating sequences (UAS) or enhancers, but the dynamic mechanisms by which activators at other sites control PIC assembly has been unclear.  In this paper, Inwha Baek (from the Buratowski lab) and Larry Friedman (from the Gelles lab) defined this mechanism using multi-wavelength single-molecule fluorescence microscopy.  The experiments used budding yeast nuclear extract with fluorescently labeled proteins and the strong artificial activator protein Gal4-VP16.  The investigators found that, unexpectedly, pol II and often TFIIE and TFIIF were not recruited directly to the promoter.  Instead they first bound via the activator to the UAS and were then subsequently transferred, likely as a pre-formed complex, to the promoter.  This work gives new insight into how messenger RNA synthesis is regulated to switch genes on and off in eukaryotic cells.  It also suggests how multiple pol II molecules may be poised at UAS sequences ready to transcribe an adjacent gene, which may explain some of the “bursts” of transcription detected in living cells.

Baek I., et al., Single-molecule studies reveal branched pathways for activator-dependent assembly of RNA polymerase II pre-initiation complexes
Molecular Cell 81, 3576-3588.e6 (2021).

DDK regulates replication initiation by controlling the multiplicity of Cdc45-GINS binding to Mcm2-7

Schematic representation of DNA replication initiation 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, Lorraine De Jesús-Kim together with collaborators from 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.”Proposed model for Cdc45-Mcm2-7-GINS (CMG) formation

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)

Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription

DNA transcription by RNA polymerase II (RNApII) is arguably the process most central to regulation of gene expression in eukaryotic organisms.  Regulated transcription requires the formation on DNA of molecular assemblies containing not only RNApII but also dozens of accessory proteins that play pivotal roles in the process.  While we know about the structures of some of these assemblies in atomic detail, quantitative understanding of the dynamics and pathways by which the assemblies interconvert and progress through this fundamental gene expression pathway is largely lacking.

In this study we report single-molecule fluorescence microscopy studies of transcription in yeast nuclear extract, for the first time visualizing and measuring the dynamics of activator-dependent recruitment of RNApII and the central elongation factor Spt4/5 to transcription complexes.  Grace Rosen (Jeff Gelles’ labortatory, Brandeis) , Inwha Baek (Steve, Buratowski’s lab, Harvard Medical School), and collaborators elucidated the kinetically significant steps in activated RNApII transcription initiation and show for the first time that Spt4/5 dynamics are tuned to the typical lifetimes of transcription elongation complexes.  In addition to these substantive results, our work represents an important methodological advance.  As the first application of the CoSMoS (co-localization single-molecule spectroscopy) technique to activated eukaryotic transcription, it demonstrates a general method for elucidating the correlated dynamic interactions of different components of the machinery with initiation and elongation transcription complexes.  The approach is likely to find further use in studies of the mechanistic features of RNApII transcription.

Rosen, G.A., Baek, I., et al., Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription