scholarly journals Action of CMG with strand-specific DNA blocks supports an internal unwinding mode for the eukaryotic replicative helicase

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Lance Langston ◽  
Mike O’Donnell
Keyword(s):  
Exclusion Process ◽  
The Other ◽  
Dna Unwinding ◽  
Duplex Dna ◽  
Central Channel ◽  
Replicative Helicase ◽  
Steric Exclusion ◽  
Dna Strand

Replicative helicases are ring-shaped hexamers that encircle DNA for duplex unwinding. The currently accepted view of hexameric helicase function is by steric exclusion, where the helicase encircles one DNA strand and excludes the other, acting as a wedge with an external DNA unwinding point during translocation. Accordingly, strand-specific blocks only affect these helicases when placed on the tracking strand, not the excluded strand. We examined the effect of blocks on the eukaryotic CMG and, contrary to expectations, blocks on either strand inhibit CMG unwinding. A recent cryoEM structure of yeast CMG shows that duplex DNA enters the helicase and unwinding occurs in the central channel. The results of this report inform important aspects of the structure, and we propose that CMG functions by a modified steric exclusion process in which both strands enter the helicase and the duplex unwinding point is internal, followed by exclusion of the non-tracking strand.

scholarly journals Mcm10 functions to isomerize CMG-DNA for replisome bypass of DNA blocks

2017 ◽  
Author(s):  
Lance D. Langston ◽  
Ryan Mayle ◽  
Grant D. Schauer ◽  
Olga Yurieva ◽  
Daniel Zhang ◽  
...  
Keyword(s):  
Cell Types ◽  
The Other ◽  
Replication Protein ◽  
Duplex Dna ◽  
Replication Forks ◽  
Central Channel ◽  
Steric Exclusion ◽  
Dna Complex ◽  
Lagging Strand

AbstractReplicative helicases of all cell types are rings that unwind DNA by steric exclusion in which the helicase ring only encircles the tracking strand, excluding the other strand outside the ring. Steric exclusion mediated unwinding enables helicase rings to bypass blocks on the strand that is excluded from the central channel. Unlike other replicative helicases, eukaryotic CMG encircles duplex DNA at a forked junction and is stopped by a block on the non-tracking (lagging) strand. This report demonstrates that Mcm10, an essential replication protein unique to eukaryotes, binds CMG and enables the replisome to bypass blocks on the non-tracking strand, implying that Mcm10 isomerizes the CMG-DNA complex to position only one strand through the central channel. A similar CMG-DNA isomerization is needed at the origin for head-to-head CMGs to bypass one another during formation of bidirectional replication forks.

scholarly journals Mcm10 promotes rapid isomerization of CMG-DNA for replisome bypass of lagging strand DNA blocks

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Lance D Langston ◽  
Ryan Mayle ◽  
Grant D Schauer ◽  
Olga Yurieva ◽  
Daniel Zhang ◽  
...  
Keyword(s):  
Cell Types ◽  
The Other ◽  
Replication Protein ◽  
Helicase Activity ◽  
Central Channel ◽  
Steric Exclusion ◽  
Dna Strand ◽  
Dna Complex ◽  
Lagging Strand

Replicative helicases in all cell types are hexameric rings that unwind DNA by steric exclusion in which the helicase encircles the tracking strand only and excludes the other strand from the ring. This mode of translocation allows helicases to bypass blocks on the strand that is excluded from the central channel. Unlike other replicative helicases, eukaryotic CMG helicase partially encircles duplex DNA at a forked junction and is stopped by a block on the non-tracking (lagging) strand. This report demonstrates that Mcm10, an essential replication protein unique to eukaryotes, binds CMG and greatly stimulates its helicase activity in vitro. Most significantly, Mcm10 enables CMG and the replisome to bypass blocks on the non-tracking DNA strand. We demonstrate that bypass occurs without displacement of the blocks and therefore Mcm10 must isomerize the CMG-DNA complex to achieve the bypass function.

A Hexameric Helicase Encircles One DNA Strand and Excludes the Other during DNA Unwinding†

Biochemistry ◽  
1997 ◽  
Vol 36 (46) ◽  
pp. 14080-14087 ◽  
Author(s):  
Kevin J. Hacker ◽  
Kenneth A. Johnson
Keyword(s):  
The Other ◽  
Dna Unwinding ◽  
Dna Strand

scholarly journals Mechanism of RPA-Facilitated Processive DNA Unwinding by the Eukaryotic CMG Helicase

2019 ◽  
Author(s):  
Hazal B. Kose ◽  
Sherry Xie ◽  
George Cameron ◽  
Melania S. Strycharska ◽  
Hasan Yardimci
Keyword(s):  
Single Molecule ◽  
Replication Fork ◽  
Double Helix ◽  
Dna Unwinding ◽  
Helicase Activity ◽  
Replicative Helicase ◽  
Double Stranded Dna ◽  
Order Of Magnitude ◽  
Dna Strand ◽  
Dna Double Helix

AbstractThe DNA double helix is unwound by the Cdc45/Mcm2-7/GINS (CMG) complex at the eukaryotic replication fork. While isolated CMG unwinds duplex DNA very slowly, its fork unwinding rate is stimulated by an order of magnitude by single-stranded DNA binding protein, RPA. However, the molecular mechanism by which RPA enhances CMG helicase activity remained elusive. Here, we demonstrate that engagement of CMG with parental double-stranded DNA (dsDNA) at the replication fork impairs its helicase activity, explaining the slow DNA unwinding by isolated CMG. Using single-molecule and ensemble biochemistry, we show that binding of RPA to the excluded DNA strand prevents duplex engagement by the helicase and speeds up CMG-mediated DNA unwinding. When stalled due to dsDNA interaction, DNA rezipping-induced helicase backtracking re-establishes productive helicase-fork engagement underscoring the significance of plasticity in helicase action. Together, our results elucidate the dynamics of CMG at the replication fork and reveal how other replisome components can mediate proper DNA engagement by the replicative helicase to achieve efficient fork progression.

scholarly journals DNA Unwinding Is an MCM Complex-dependent and ATP Hydrolysis-dependent Process

2004 ◽  
Vol 279 (44) ◽  
pp. 45586-45593 ◽  
Author(s):  
David Shechter ◽  
Carol Y. Ying ◽  
Jean Gautier
Keyword(s):  
Dna Replication ◽  
Neutralizing Antibodies ◽  
Atp Hydrolysis ◽  
Initial Period ◽  
Dna Helicase ◽  
Dna Unwinding ◽  
Origin Firing ◽  
Replicative Helicase ◽  
Mcm Proteins

Minichromosome maintenance proteins (Mcm) are essential in all eukaryotes and are absolutely required for initiation of DNA replication. The eukaryotic and archaeal Mcm proteins have conserved helicase motifs and exhibit DNA helicase and ATP hydrolysis activitiesin vitro. Although the Mcm proteins have been proposed to be the replicative helicase, the enzyme that melts the DNA helix at the replication fork, their function during cellular DNA replication elongation is still unclear. Using nucleoplasmic extract (NPE) fromXenopus laeviseggs and six purified polyclonal antibodies generated against each of theXenopusMcm proteins, we have demonstrated that Mcm proteins are required during DNA replication and DNA unwinding after initiation of replication. Quantitative depletion of Mcms from the NPE results in normal replication and unwinding, confirming that Mcms are required before pre-replicative complex assembly and dispensable thereafter. Replication and unwinding are inhibited when pooled neutralizing antibodies against the six different Mcm2–7 proteins are added during NPE incubation. Furthermore, replication is blocked by the addition of the Mcm antibodies after an initial period of replication in the NPE, visualized by a pulse of radiolabeled nucleotide at the same time as antibody addition. Addition of the cyclin-dependent kinase 2 inhibitor p21cip1specifically blocks origin firing but does not prevent helicase action. When p21cip1is added, followed by the non-hydrolyzable analog ATPγS to block helicase function, unwinding is inhibited, demonstrating that plasmid unwinding is specifically attributable to an ATP hydrolysis-dependent function. These data support the hypothesis that the Mcm protein complex functions as the replicative helicase.

scholarly journals A DNA-binding protein from Ustilago maydis prefers duplex DNA without chain interruptions.

1983 ◽  
Vol 3 (4) ◽  
pp. 605-612 ◽  
Author(s):  
J R Rusche ◽  
W K Holloman
Keyword(s):  
Drosophila Melanogaster ◽  
Satellite Dna ◽  
Binding Assay ◽  
Ustilago Maydis ◽  
Dna Binding Protein ◽  
Double Strand Breaks ◽  
Duplex Dna ◽  
Plasmid Pbr322 ◽  
Strand Breaks ◽  
Dna Strand

Using a nitrocellulose filter binding assay, we have partially purified a protein from mitotic cells of Ustilago maydis that binds preferentially to covalently closed circular duplex DNA. DNA containing single- or double-strand breaks is bound poorly by the protein. Once formed, the DNA-protein complex is stable, resisting dissociation in high salt. However, when a DNA strand is broken, the complex appears to dissociate. The protein binds equally well to form I DNA of phi X174 or the plasmid pBR322, but has a higher affinity for a hybrid plasmid containing a cloned region of Drosophila melanogaster satellite DNA.

scholarly journals Duplex DNA engagement and RPA oppositely regulate the DNA-unwinding rate of CMG helicase

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Hazal B. Kose ◽  
Sherry Xie ◽  
George Cameron ◽  
Melania S. Strycharska ◽  
Hasan Yardimci
Keyword(s):  
Dna Unwinding ◽  
Duplex Dna

Multidetectors for Analysis of the Composition of Copolymers as a Function of Molecular Size Distribution by Steric Exclusion Chromatography

1973 ◽  
Vol 46 (2) ◽  
pp. 449-463 ◽  
Author(s):  
D. J. Harmon ◽  
V. L. Folt
Keyword(s):  
Liquid Chromatography ◽  
Size Distribution ◽  
Oil Content ◽  
Molecular Size ◽  
The Other ◽  
Additional Information ◽  
Steric Exclusion ◽  
Gel Permeation ◽  
Exclusion Chromatography ◽  
Molecular Size Distribution

Abstract Analysis of molecular size distribution of polymers by steric exclusion liquid chromatography (GPC) is well known. Problems exist, however. These problems involve copolymers and polymer blends. The objectives of the research were to develop methods of analyzing comonomer distribution in copolymers, to study the breakdown of one polymer independent of another in a polymer blend, and to obtain any additional information as might be available. The separations were performed on a Waters Model 200 Gel Permeation Chromatograph. Detectors employed were a Waters R-4 differential refractometer, a Wilks Miran-1 infrared analyzer, and a Beckman Model 144 UV photometer. Examples are given of analysis of average styrene, styrene distribution, and oil content of oil extended SBR. The data is compared with that obtained by other methods. In general the agreement is good. The ability to examine one polymer of a blend independent of the other is also demonstrated. Since elastomers are frequently used as blends, this becomes very important to such studies as milling and extrusion behavior.

scholarly journals RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes

2016 ◽  
Vol 113 (52) ◽  
pp. 14994-14999 ◽  
Author(s):  
Monali NandyMazumdar ◽  
Yuri Nedialkov ◽  
Dmitri Svetlov ◽  
Anastasia Sevostyanova ◽  
Georgiy A. Belogurov ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Elongation Factor ◽  
Structural Data ◽  
Duplex Dna ◽  
Structural Element ◽  
Transcript Cleavage ◽  
Σ Factor ◽  
Transcription Complexes ◽  
Dna Strand ◽  
Many Core

Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β′ clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.

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