Tag Archives: yeast

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

 

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)

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.

https://doi.org/10.1073/pnas.2011224117
Rosen, G.A., Baek, I., et al., Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription
PNAS