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Replication origins are licensed by loading two Mcm2-7 helicases around DNA in a head-to-head conformation poised to initiate bidirectional replication. This process requires origin–recognition complex (ORC), Cdc6, and Cdt1. Although different Cdc6 and Cdt1 molecules load each helicase, whether two ORC proteins are required is unclear. Using colocalization single-molecule spectroscopy combined with single-molecule Förster resonance energy transfer (FRET), we investigated interactions between ORC and Mcm2-7 during helicase loading. In the large majority of events, we observed a single ORC molecule recruiting both Mcm2-7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between first- and second-helicase recruitment, a rapid change in interactions between ORC and the first Mcm2-7 occurs. Within seconds, ORC breaks the interactions mediating first Mcm2-7 recruitment, releases from its initial DNA-binding site, and forms a new interaction with the opposite face of the first Mcm2-7. This rearrangement requires release of the first Cdt1 and tethers ORC as it flips over the first Mcm2-7 to form an inverted Mcm2-7–ORC–DNA complex required for second-helicase recruitment. To ensure correct licensing, this complex is maintained until head-to-head interactions between the two helicases are formed. Our findings reconcile previous observations and reveal a highly coordinated series of events through which a single ORC molecule can load two oppositely oriented helicases.

© 2020, The Author(s). RNA polymerases (RNAPs) transcribe genes through a cycle of recruitment to promoter DNA, initiation, elongation, and termination. After termination, RNAP is thought to initiate the next round of transcription by detaching from DNA and rebinding a new promoter. Here we use single-molecule fluorescence microscopy to observe individual RNAP molecules after transcript release at a terminator. Following termination, RNAP almost always remains bound to DNA and sometimes exhibits one-dimensional sliding over thousands of basepairs. Unexpectedly, the DNA-bound RNAP often restarts transcription, usually in reverse direction, thus producing an antisense transcript. Furthermore, we report evidence of this secondary initiation in live cells, using genome-wide RNA sequencing. These findings reveal an alternative transcription cycle that allows RNAP to reinitiate without dissociating from DNA, which is likely to have important implications for gene regulation.

© 2021, eLife Sciences Publications Ltd. All rights reserved. The committed step of eukaryotic DNA replication occurs when the pairs of Mcm2-7 replicative helicases that license each replication origin are activated. Helicase activation requires the recruitment of Cdc45 and GINS to Mcm2-7, forming Cdc45-Mcm2-7-GINS complexes (CMGs). Using single-molecule biochemical assays to monitor CMG formation, we found that Cdc45 and GINS are recruited to loaded Mcm2-7 in two stages. Initially, Cdc45, GINS, and likely additional proteins are recruited to unstructured Mcm2-7 N-terminal tails in a Dbf4-dependent kinase (DDK)-dependent manner, forming Cdc45-tail-GINS intermediates (CtGs). DDK phosphorylation of multiple phosphorylation sites on the Mcm2-7 tails modulates the number of CtGs formed per Mcm2-7. In a second, inefficient event, a subset of CtGs transfer their Cdc45 and GINS components to form CMGs. Importantly, higher CtG multiplicity increases the frequency of CMG formation. Our findings reveal molecular mechanisms sensitizing helicase activation to DDK levels with implications for control of replication origin efficiency and timing.

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. To understand the formation and importance of this double-hexamer, we identified mutations in a conserved and essential McM4 motif that permit loading of two McM2-7 complexes but are defective for double-hexamer formation. Single-molecule studies show mutant McM2-7 forms initial hexamer-hexamer interactions; however, the resulting complex is unstable. Kinetic analyses of wild-type and mutant McM2-7 reveal a limited time window for double-hexamer formation following second McM2-7 association, suggesting that this process is facilitated. Double-hexamer formation is required for extensive origin DNA unwinding but not initial DNA melting or recruitment of helicase-activation proteins (Cdc45, GINS, McM10). Our findings elucidate dynamic mechanisms of origin licensing, and identify the transition between initial DNA melting and extensive unwinding as the first initiation event requiring double-hexamer formation., National Institute of General Medical Sciences (Grant GM52339), National Cancer Institute (Grant P30-CA14051)

The opening and closing of two ring-shaped Mcm2-7 DNA helicases is necessary to license eukaryotic origins of replication, although the mechanisms controlling these events are unclear. The origin-recognition complex (ORC), Cdc6 and Cdt1 facilitate this process by establishing a topological link between each Mcm2-7 hexamer and origin DNA. Using colocalization single-molecule spectroscopy and single-molecule Förster resonance energy transfer (FRET), we monitored ring opening and closing of Saccharomyces cerevisiae Mcm2-7 during origin licensing. The two Mcm2-7 rings were open during initial DNA association and closed sequentially, concomitant with the release of their associated Cdt1. We observed that ATP hydrolysis by Mcm2-7 was coupled to ring closure and Cdt1 release, and failure to load the first Mcm2-7 prevented recruitment of the second Mcm2-7. Our findings identify key mechanisms controlling the Mcm2-7 DNA-entry gate during origin licensing, and reveal that the two Mcm2-7 complexes are loaded via a coordinated series of events with implications for bidirectional replication initiation and quality control., National Institutes of Health (U.S.) (Grant R01 GM52339), National Institutes of Health (U.S.) (Pre-Doctoral Training Grant GM007287), National Cancer Institute (U.S.) (Koch Institute Support Grant P30-CA14051)

Loading of the ring-shaped Mcm2–7 replicative helicase around DNA licenses eukaryotic origins of replication. During loading, Cdc6, Cdt1, and the origin-recognition complex (ORC) assemble two heterohexameric Mcm2–7 complexes into a head-to-head double hexamer that facilitates bidirectional replication initiation. Using multi-wavelength single-molecule fluorescence to monitor the events of helicase loading, we demonstrate that double-hexamer formation is the result of sequential loading of individual Mcm2–7 complexes. Loading of each Mcm2–7 molecule involves the ordered association and dissociation of distinct Cdc6 and Cdt1 proteins. In contrast, one ORC molecule directs loading of both helicases in each double hexamer. Based on single-molecule FRET, arrival of the second Mcm2–7 results in rapid double-hexamer formation that anticipates Cdc6 and Cdt1 release, suggesting that Mcm-Mcm interactions recruit the second helicase. Our findings reveal the complex protein dynamics that coordinate helicase loading and indicate that distinct mechanisms load the oppositely oriented helicases that are central to bidirectional replication initiation., National Institutes of Health (U.S.) (NIH grant GM52339), National Institutes of Health (U.S.) (NIH grant R01 GM81648), G. Harold and Leila Y. Mathers Foundation, National Institutes of Health (U.S.) (NIH Pre-Doctoral Training Grant (GM007287)), Howard Hughes Medical Institute (Investigator)

A canonical quantitative view of transcriptional regulation holds that the only role of operator sequence is to set the probability of transcription factor binding, with operator occupancy determining the level of gene expression. In this work, we test this idea by characterizing repression in vivo and the binding of RNA polymerase in vitro in experiments where operators of various sequences were placed either upstream or downstream from the promoter in Escherichia coli. Surprisingly, we find that operators with a weaker binding affinity can yield higher repression levels than stronger operators. Repressor bound to upstream operators modulates promoter escape, and the magnitude of this modulation is not correlated with the repressor-operator binding affinity. This suggests that operator sequences may modulate transcription by altering the nature of the interaction of the bound transcription factor with the transcriptional machinery, implying a new layer of sequence dependence that must be confronted in the quantitative understanding of gene expression., National Institutes of Health (U.S.) (Grant GM81648), National Institutes of Health (U.S.) (Grant GM43369)

Cytochrome c oxidase contains a copper-ion electron transfer site, CuA, which has previously been found to be unreactive with externally added reagents under conditions in which the protein remains structurally intact. We have studied the reaction of cytochrome oxidase with sodium p-hydroxymercuribenzoate (pHMB) and found that the reaction proceeds, under appropriate conditions, to give an excellent yield of a particular derivative of the CuA center which has electron paramagnetic resonance and near-infrared absorption spectroscopic properties which are distinctly different from those of the unmodified center. Spectroscopic and chemical characterization of the other metal-ion sites of the enzyme reveals little or no effect of the pHMB modification on the structures of and reactions at those sites. Of particular interest is the observation that the modified enzyme still displays a substantial fraction of the native steady-state activity of electron transfer from ferrocytochrome c to O2. Although the modified copper center retains the ability to receive electrons from the powerful reductant Na2S2O4 and to transfer electrons to O2, it is not significantly reduced when the enzyme is treated with milder (higher potential) reductants such as NADH/phenazine methosulfate or the physiological substrate ferrocytochrome c. CuA exhibits many spectroscopic and chemical properties which make it highly atypical of cuproprotein active sites; the singular nature of this site has prompted speculation about the importance of the structural peculiarities of this metal-ion center in the catalytic cycle of the enzyme. In this work, we demonstrate that the unusual features of this site are not prerequisites for competent catalysis of electron transfer and O2 reduction by the enzyme. These observations suggest that a secondary electron transfer pathway not involving CuA e

Cytochrome c oxidase contains a copper-ion electron transfer site, CuA, which has previously been found to be unreactive with externally added reagents under conditions in which the protein remains structurally intact. We have studied the reaction of cytochrome oxidase with sodium p-hydroxymercuribenzoate (pHMB) and found that the reaction proceeds, under appropriate conditions, to give an excellent yield of a particular derivative of the CuA center which has electron paramagnetic resonance and near-infrared absorption spectroscopic properties which are distinctly different from those of the unmodified center. Spectroscopic and chemical characterization of the other metal-ion sites of the enzyme reveals little or no effect of the pHMB modification on the structures of and reactions at those sites. Of particular interest is the observation that the modified enzyme still displays a substantial fraction of the native steady-state activity of electron transfer from ferrocytochrome c to O2. Although the modified copper center retains the ability to receive electrons from the powerful reductant Na2S2O4 and to transfer electrons to O2, it is not significantly reduced when the enzyme is treated with milder (higher potential) reductants such as NADH/phenazine methosulfate or the physiological substrate ferrocytochrome c. CuA exhibits many spectroscopic and chemical properties which make it highly atypical of cuproprotein active sites; the singular nature of this site has prompted speculation about the importance of the structural peculiarities of this metal-ion center in the catalytic cycle of the enzyme. In this work, we demonstrate that the unusual features of this site are not prerequisites for competent catalysis of electron transfer and O2 reduction by the enzyme. These observations suggest that a secondary electron transfer pathway not involving CuA e

Cytochrome c oxidase contains a copper-ion electron transfer site, CuA, which has previously been found to be unreactive with externally added reagents under conditions in which the protein remains structurally intact. We have studied the reaction of cytochrome oxidase with sodium p-hydroxymercuribenzoate (pHMB) and found that the reaction proceeds, under appropriate conditions, to give an excellent yield of a particular derivative of the CuA center which has electron paramagnetic resonance and near-infrared absorption spectroscopic properties which are distinctly different from those of the unmodified center. Spectroscopic and chemical characterization of the other metal-ion sites of the enzyme reveals little or no effect of the pHMB modification on the structures of and reactions at those sites. Of particular interest is the observation that the modified enzyme still displays a substantial fraction of the native steady-state activity of electron transfer from ferrocytochrome c to O2. Although the modified copper center retains the ability to receive electrons from the powerful reductant Na2S2O4 and to transfer electrons to O2, it is not significantly reduced when the enzyme is treated with milder (higher potential) reductants such as NADH/phenazine methosulfate or the physiological substrate ferrocytochrome c. CuA exhibits many spectroscopic and chemical properties which make it highly atypical of cuproprotein active sites; the singular nature of this site has prompted speculation about the importance of the structural peculiarities of this metal-ion center in the catalytic cycle of the enzyme. In this work, we demonstrate that the unusual features of this site are not prerequisites for competent catalysis of electron transfer and O2 reduction by the enzyme. These observations suggest that a secondary electron transfer pathway not involving CuA e