scholarly journals The Sulfolobus solfataricus GINS Complex Stimulates DNA Binding and Processive DNA Unwinding by Minichromosome Maintenance Helicase

2015 ◽  
Vol 197 (21) ◽  
pp. 3409-3420 ◽  
Author(s):  
Shiwei Lang ◽  
Li Huang
Keyword(s):  
Dna Binding ◽  
Atpase Activity ◽  
Replication Fork ◽  
Molecular Motor ◽  
Sulfolobus Solfataricus ◽  
Dna Unwinding ◽  
Helicase Activity ◽  
Minichromosome Maintenance ◽  
Content Type ◽  
Double Stranded Dna

ABSTRACTGINS is a key component of the eukaryotic Cdc45-minichromosome maintenance (MCM)-GINS (CMG) complex, which unwinds duplex DNA at the moving replication fork. Archaeal GINS complexes have been shown to stimulate the helicase activity of their cognate MCM mainly by elevating its ATPase activity. Here, we report that GINS from the thermoacidophilic crenarchaeonSulfolobus solfataricus(SsoGINS) is capable of DNA binding and binds preferentially to single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA). Notably, SsoGINS binds more strongly to dsDNA with a 5′ ssDNA tail than to dsDNA with a 3′ tail and more strongly to an ssDNA fragment blocked at the 3′ end than to one at the 5′ end with a biotin-streptavidin (SA) complex, suggesting the ability of the protein complex to slide in a 5′-to-3′ direction along ssDNA. DNA-bound SsoGINS enhances DNA binding by SsoMCM. Furthermore, SsoGINS increases the helicase activity of SsoMCM. However, the ATPase activity of SsoMCM is not affected by SsoGINS. Our results suggest that SsoGINS facilitates processive DNA unwinding by SsoMCM by enhancing the binding of the helicase to DNA. We propose that SsoGINS stabilizes the interaction of SsoMCM with the replication fork and moves along with the helicase as the fork progresses.IMPORTANCEGINS is a key component of the eukaryotic Cdc45-MCM-GINS complex, a molecular motor that drives the unwinding of DNA in front of the replication fork.Archaeaalso encode GINS, which interacts with MCM, the helicase. But how archaeal GINS serves its role remains to be understood. In this study, we show that GINS from the hyperthermophilic archaeonSulfolobus solfataricusis able to bind to DNA and slide along ssDNA in a 5′-to-3′ direction. Furthermore,SulfolobusGINS enhances DNA binding by MCM, which slides along ssDNA in a 3′-to-5′ direction. Taken together, these results suggest thatSulfolobusGINS may stabilize the interaction of MCM with the moving replication fork, facilitating processive DNA unwinding.

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 Binding by the Methanothermobacter thermautotrophicus Cdc6 Protein Is Inhibited by the Minichromosome Maintenance Helicase

2006 ◽  
Vol 188 (12) ◽  
pp. 4577-4580 ◽  
Author(s):  
Rajesh Kasiviswanathan ◽  
Jae-Ho Shin ◽  
Zvi Kelman
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Minichromosome Maintenance ◽  
Single Stranded Dna ◽  
Mutant Proteins ◽  
Dna Binding Activity ◽  
Double Stranded Dna ◽  
Mcm Helicase ◽  
Methanothermobacter Thermautotrophicus

ABSTRACT The Cdc6 proteins from the archaeon Methanothermobacter thermautotrophicus were previously shown to bind double-stranded DNA. It is shown here that the proteins also bind single-stranded DNA. Using minichromosome maintenance (MCM) helicase mutant proteins unable to bind DNA, it was found that the interaction of MCM with Cdc6 inhibits the DNA binding activity of Cdc6.

scholarly journals The interplay of DNA binding, ATP hydrolysis and helicase activities of the archaeal MCM helicase

2011 ◽  
Vol 436 (2) ◽  
pp. 409-414 ◽  
Author(s):  
Li Phing Liew ◽  
Stephen D. Bell
Keyword(s):  
Dna Binding ◽  
Active Site ◽  
Atp Hydrolysis ◽  
Sulfolobus Solfataricus ◽  
Active Form ◽  
Dna Helicase ◽  
Atpase Domain ◽  
Minichromosome Maintenance ◽  
Conserved Residues ◽  
Mcm Helicase

The MCM (minichromosome maintenance) proteins of archaea are widely believed to be the replicative DNA helicase of these organisms. Most archaea possess a single MCM orthologue that forms homo-multimeric assemblies with a single hexamer believed to be the active form. In the present study we characterize the roles of highly conserved residues in the ATPase domain of the MCM of the hyperthermophilic archaeon Sulfolobus solfataricus. Our results identify a potential conduit for communicating DNA-binding information to the ATPase active site.

scholarly journals Parvovirus Initiator Protein NS1 and RPA Coordinate Replication Fork Progression in a Reconstituted DNA Replication System

2002 ◽  
Vol 76 (13) ◽  
pp. 6518-6531 ◽  
Author(s):  
Jesper Christensen ◽  
Peter Tattersall
Keyword(s):  
Dna Binding ◽  
Replication Fork ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Small Subunit ◽  
Dna Helicase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Helicase Activity ◽  
Single Stranded Dna ◽  
Initiator Protein

ABSTRACT We show here that the DNA helicase activity of the parvoviral initiator protein NS1 is highly directional, binding to the single strand at a recessed 5′ end and displacing the other strand while progressing in a 3′-to-5′ direction on the bound strand. NS1 and a cellular site-specific DNA binding factor, PIF, also known as glucocorticoid modulating element binding protein, bind to the left-end minimal replication origin of minute virus of mice, forming a ternary complex. In this complex, NS1 is activated to nick one DNA strand, becoming covalently attached to the 5′ end of the nick in the process and providing a 3′ OH for priming DNA synthesis. In this situation, the helicase activity of NS1 did not displace the nicked strand, but the origin duplex was distorted by the NS1-PIF complex, as assayed by its sensitivity to KMnO4 oxidation, and a stretch of about 14 nucleotides on both strands of the nicked origin underwent limited unwinding. Addition of Escherichia coli single-stranded DNA binding protein (SSB) did not lead to further unwinding. However, addition of recombinant human single-stranded DNA binding protein (RPA) to the initiation reaction catalyzed extensive unwinding of the nicked origin, suggesting that RPA may be required to form a functional replication fork. Accordingly, the unwinding mediated by NS1 and RPA promoted processive leading-strand synthesis catalyzed by recombinant human DNA polymerase δ, PCNA, and RFC, using the minimal left-end origin cloned in a plasmid as a template. The requirement for RPA, rather than SSB, in the unwinding reaction indicated that specific NS1-RPA protein interactions were formed. NS1 was tested by enzyme-linked immunosorbent assay for binding to two- or three-subunit RPA complexes expressed from recombinant baculoviruses. NS1 efficiently bound each of the baculovirus-expressed complexes, indicating that the small subunit of RPA is not involved in specific NS1 binding. No NS1 interactions were observed with E. coli SSB or other proteins included as controls.

scholarly journals Domain Organization of Vaccinia Virus Helicase-Primase D5

2016 ◽  
Vol 90 (9) ◽  
pp. 4604-4613 ◽  
Author(s):  
Stephanie Hutin ◽  
Wai Li Ling ◽  
Adam Round ◽  
Gregory Effantin ◽  
Stefan Reich ◽  
...  
Keyword(s):  
Dna Binding ◽  
Vaccinia Virus ◽  
Genome Structure ◽  
Antiviral Drug ◽  
Binding Activity ◽  
Structural Basis ◽  
Helicase Domain ◽  
Content Type ◽  
Dna Binding Activity ◽  
Double Stranded Dna

ABSTRACTPoxviridaeare viruses with a large linear double-stranded DNA genome coding for up to 250 open reading frames and a fully cytoplasmic replication. The double-stranded DNA genome is covalently circularized at both ends. Similar structures of covalently linked extremities of the linear DNA genome are found in the African swine fever virus (asfarvirus) and in thePhycodnaviridae. We are studying the machinery which replicates this peculiar genome structure. From our work with vaccinia virus, we give first insights into the overall structure and function of the essential poxvirus virus helicase-primase D5 and show that the active helicase domain of D5 builds a hexameric ring structure. This hexamer has ATPase and, more generally, nucleoside triphosphatase activities that are indistinguishable from the activities of full-length D5 and that are independent of the nature of the base. In addition, hexameric helicase domains bind tightly to single- and double-stranded DNA. Still, the monomeric D5 helicase construct truncated within the D5N domain leads to a well-defined structure, but it does not have ATPase or DNA-binding activity. This shows that the full D5N domain has to be present for hexamerization. This allowed us to assign a function to the D5N domain which is present not only in D5 but also in other viruses of the nucleocytoplasmic large DNA virus (NCLDV) clade. The primase domain and the helicase domain were structurally analyzed via a combination of small-angle X-ray scattering and, when appropriate, electron microscopy, leading to consistent low-resolution models of the different proteins.IMPORTANCESince the beginning of the 1980s, research on the vaccinia virus replication mechanism has basically stalled due to the absence of structural information. As a result, this important class of pathogens is less well understood than most other viruses. This lack of information concerns in general viruses of the NCLDV clade, which use a superfamily 3 helicase for replication, as do poxviruses. Here we provide for the first time information about the domain structure and DNA-binding activity of D5, the poxvirus helicase-primase. This result not only refines the current model of the poxvirus replication fork but also will lead in the long run to a structural basis for antiviral drug design.

scholarly journals An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor

2015 ◽  
Vol 89 (24) ◽  
pp. 12457-12466 ◽  
Author(s):  
Wei Zhao ◽  
Paul J. Jardine ◽  
Shelley Grimes
Keyword(s):  
Viral Genome ◽  
Virus Assembly ◽  
Molecular Motor ◽  
Protein A ◽  
Dna Packaging ◽  
Viral Dna ◽  
High Biological Activity ◽  
Content Type ◽  
Double Stranded Dna

ABSTRACTDuring assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in theBacillus subtilisbacteriophage ϕ29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activityin vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of ϕ29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although ϕ29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages.IMPORTANCEDuring virus assembly, genome packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful molecular motor that translocates the viral DNA into a preformed viral shell. A key event in DNA packaging is recognition of the viral DNA among other nucleic acids in the host cell. Commonly, a DNA-binding protein mediates the interaction of viral DNA with the motor/head shell. Here we show that for the bacteriophage ϕ29, this essential step of genome recognition is mediated by a viral genome-encoded RNA rather than a protein. A domain of the prohead RNA (pRNA) imparts specificity and stringency to the motor by ensuring the correct orientation of DNA packaging and restricting initiation to a single event. Since this assembly step is unique to the virus, DNA packaging is a novel target for the development of antiviral drugs.

scholarly journals The eukaryotic replisome requires an additional helicase to disarm dormant replication origins

2020 ◽  
Author(s):  
Jake Hill ◽  
Patrik Eickhoff ◽  
Lucy S. Drury ◽  
Alessandro Costa ◽  
John F.X. Diffley
Keyword(s):  
Replication Fork ◽  
S Phase ◽  
Replication Forks ◽  
Minichromosome Maintenance ◽  
Double Stranded Dna ◽  
Efficient Replication ◽  
Eukaryotic Dna ◽  
Mcm Proteins ◽  
Fork Stalling ◽  
Pif1 Helicase

Origins of eukaryotic DNA replication are ‘licensed’ during G1 phase of the cell cycle by loading the six related minichromosome maintenance (MCM) proteins into a double hexameric ring around double-stranded DNA. In S phase, some double hexamers (MCM DHs) are converted into active CMG (Cdc45-MCM-GINS) helicases which nucleate assembly of bidirectional replication forks. The remaining unfired MCM DHs act as ‘dormant’ origins to provide backup replisomes in the event of replication fork stalling. The fate of unfired MCM DHs during replication is unknown. Here we show that active replisomes cannot remove unfired MCM DHs. Instead, they are pushed ahead of the replisome where they prevent fork convergence during replication termination and replisome progression through nucleosomes. Pif1 helicase, together with the replisome, can remove unfired MCM DHs specifically from replicating DNA, allowing efficient replication and termination. Our results provide an explanation for how excess replication license is removed during S phase.

scholarly journals The eukaryotic replisome requires an additional helicase to disarm dormant replication origins

2020 ◽  
Author(s):  
Jake Hill ◽  
Patrik Eickhoff ◽  
Lucy Drury ◽  
Alessandro Costa ◽  
John Diffley
Keyword(s):  
Replication Fork ◽  
S Phase ◽  
Replication Forks ◽  
Minichromosome Maintenance ◽  
Double Stranded Dna ◽  
Efficient Replication ◽  
Eukaryotic Dna ◽  
Mcm Proteins ◽  
Fork Stalling ◽  
Pif1 Helicase

Abstract Origins of eukaryotic DNA replication are ‘licensed’ during G1 phase of the cell cycle by loading the six related minichromosome maintenance (MCM) proteins into a double hexameric ring around double-stranded DNA. In S phase, some double hexamers (MCM DHs) are converted into active CMG (Cdc45-MCM-GINS) helicases which nucleate assembly of bidirectional replication forks. The remaining unfired MCM DHs act as ‘dormant’ origins to provide backup replisomes in the event of replication fork stalling. The fate of unfired MCM DHs during replication is unknown. Here we show that active replisomes cannot remove unfired MCM DHs. Instead, they are pushed ahead of the replisome where they prevent fork convergence during replication termination and replisome progression through nucleosomes. Pif1 helicase, together with the replisome, can remove unfired MCM DHs specifically from replicating DNA, allowing efficient replication and termination. Our results provide an explanation for how excess replication license is removed during S phase.

scholarly journals Replication Fork Breakage and Restart inEscherichia coli

2018 ◽  
Vol 82 (3) ◽  
Author(s):  
Bénédicte Michel ◽  
Anurag K. Sinha ◽  
David R. F. Leach
Keyword(s):  
Replication Fork ◽  
Strand Break ◽  
Replication Forks ◽  
Wild Type ◽  
Dsb Repair ◽  
Replicative Helicase ◽  
Content Type ◽  
E Coli ◽  
Double Stranded Dna ◽  
Replication Restart

SUMMARYIn all organisms, replication impairments are an important source of genome rearrangements, mainly because of the formation of double-stranded DNA (dsDNA) ends at inactivated replication forks. Three reactions for the formation of dsDNA ends at replication forks were originally described forEscherichia coliand became seminal models for all organisms: the encounter of replication forks with preexisting single-stranded DNA (ssDNA) interruptions, replication fork reversal, and head-to-tail collisions of successive replication rounds. Here, we first review the experimental evidence that now allows us to know when, where, and how these three different reactions occur inE. coli. Next, we recall our recent studies showing that in wild-typeE. coli, spontaneous replication fork breakage occurs in 18% of cells at each generation. We propose that it results from the replication of preexisting nicks or gaps, since it does not involve replication fork reversal or head-to-tail fork collisions. In therecBmutant, deficient for double-strand break (DSB) repair, fork breakage triggers DSBs in the chromosome terminus during cell division, a reaction that is heritable for several generations. Finally, we recapitulate several observations suggesting that restart from intact inactivated replication forks and restart from recombination intermediates require different sets of enzymatic activities. The finding that 18% of cells suffer replication fork breakage suggests that DNA remains intact at most inactivated forks. Similarly, only 18% of cells need the helicase loader for replication restart, which leads us to speculate that the replicative helicase remains on DNA at intact inactivated replication forks and is reactivated by the replication restart proteins.

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