scholarly journals A conserved MCM single-stranded DNA binding element is essential for replication initiation

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Clifford A Froelich ◽  
Sukhyun Kang ◽  
Leslie B Epling ◽  
Stephen P Bell ◽  
Eric J Enemark
Keyword(s):  
Pyrococcus Furiosus ◽  
Dna Helicase ◽  
Replication Initiation ◽  
Binding Motif ◽  
Replication Origins ◽  
Dna Interactions ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Helicase Loading ◽  
Mcm Helicase

The ring-shaped MCM helicase is essential to all phases of DNA replication. The complex loads at replication origins as an inactive double-hexamer encircling duplex DNA. Helicase activation converts this species to two active single hexamers that encircle single-stranded DNA (ssDNA). The molecular details of MCM DNA interactions during these events are unknown. We determined the crystal structure of the Pyrococcus furiosus MCM N-terminal domain hexamer bound to ssDNA and define a conserved MCM-ssDNA binding motif (MSSB). Intriguingly, ssDNA binds the MCM ring interior perpendicular to the central channel with defined polarity. In eukaryotes, the MSSB is conserved in several Mcm2-7 subunits, and MSSB mutant combinations in S. cerevisiae Mcm2-7 are not viable. Mutant Mcm2-7 complexes assemble and are recruited to replication origins, but are defective in helicase loading and activation. Our findings identify an important MCM-ssDNA interaction and suggest it functions during helicase activation to select the strand for translocation.

scholarly journals Analysis of the crystal structure of an active MCM hexamer

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Justin M Miller ◽  
Buenafe T Arachea ◽  
Leslie B Epling ◽  
Eric J Enemark
Keyword(s):  
Crystal Structure ◽  
Atp Hydrolysis ◽  
Pyrococcus Furiosus ◽  
Binding Pocket ◽  
Activation Mechanism ◽  
Binding Motif ◽  
Atpase Domain ◽  
Ssdna Binding ◽  
Allosteric Communication ◽  
Mcm Helicase

In a previous Research article (<xref ref-type="bibr" rid="bib25">Froelich et al., 2014</xref>), we suggested an MCM helicase activation mechanism, but were limited in discussing the ATPase domain because it was absent from the crystal structure. Here we present the crystal structure of a nearly full-length MCM hexamer that is helicase-active and thus has all features essential for unwinding DNA. The structure is a chimera of Sulfolobus solfataricus N-terminal domain and Pyrococcus furiosus ATPase domain. We discuss three major findings: 1) a novel conformation for the A-subdomain that could play a role in MCM regulation; 2) interaction of a universally conserved glutamine in the N-terminal Allosteric Communication Loop with the AAA+ domain helix-2-insert (h2i); and 3) a recessed binding pocket for the MCM ssDNA-binding motif influenced by the h2i. We suggest that during helicase activation, the h2i clamps down on the leading strand to facilitate strand retention and regulate ATP hydrolysis.

scholarly journals Investigating the role of Rts1 in DNA replication initiation

2018 ◽  
Vol 3 ◽  
pp. 23 ◽  
Author(s):  
Ana B.A. Wallis ◽  
Conrad A. Nieduszynski
Keyword(s):  
Dna Replication ◽  
Temperature Sensitivity ◽  
Protein Phosphatase ◽  
Dna Helicase ◽  
Replication Initiation ◽  
Replication Origins ◽  
Origin Firing ◽  
Replication Factor ◽  
Dna Replication Initiation ◽  
Genomic Screen

Background: Understanding DNA replication initiation is essential to understand the mis-regulation of replication seen in cancer and other human disorders. DNA replication initiates from DNA replication origins. In eukaryotes, replication is dependent on cell cycle kinases which function during S phase. Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) act to phosphorylate the DNA helicase (composed of mini chromosome maintenance proteins: Mcm2-7) and firing factors to activate replication origins. It has recently been found that Rif1 can oppose DDK phosphorylation. Rif1 can recruit protein phosphatase 1 (PP1) to dephosphorylate MCM and restricts origin firing. In this study, we investigate a potential role for another phosphatase, protein phosphatase 2A (PP2A), in regulating DNA replication initiation. The PP2A regulatory subunit Rts1 was previously identified in a large-scale genomic screen to have a genetic interaction with ORC2 (a DNA replication licensing factor). Deletion of RTS1 synthetically rescued the temperature-sensitive (ts-) phenotype of ORC2 mutants. Methods: We deleted RTS1 in multiple ts-replication factor Saccharomyces cerevisiae strains, including ORC2.  Dilution series assays were carried out to compare qualitatively the growth of double mutant ∆rts1 ts-replication factor strains relative to the respective single mutant strains.   Results: No synthetic rescue of temperature-sensitivity was observed. Instead we found an additive phenotype, indicating gene products function in separate biological processes. These findings are in agreement with a recent genomic screen which found that RTS1 deletion in several ts-replication factor strains led to increased temperature-sensitivity. Conclusions: We find no evidence that Rts1 is involved in the dephosphorylation of DNA replication initiation factors.

scholarly journals Identification and Characterization of the Fourth Single-Stranded-DNA Binding Domain of Replication Protein A

1998 ◽  
Vol 18 (12) ◽  
pp. 7225-7234 ◽  
Author(s):  
Steven J. Brill ◽  
Suzanne Bastin-Shanower
Keyword(s):  
Protein A ◽  
Replication Protein A ◽  
Binding Domain ◽  
Replication Protein ◽  
Binding Motif ◽  
Terminal Portion ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Amino Terminal ◽  
Binding Domains

ABSTRACT Replication protein A (RPA), the heterotrimeric single-stranded-DNA (ssDNA) binding protein (SSB) of eukaryotes, contains two homologous ssDNA binding domains (A and B) in its largest subunit, RPA1, and a third domain in its second-largest subunit, RPA2. Here we report that Saccharomyces cerevisiae RPA1 contains a previously undetected ssDNA binding domain (domain C) lying in tandem with domains A and B. The carboxy-terminal portion of domain C shows sequence similarity to domains A and B and to the region of RPA2 that binds ssDNA (domain D). The aromatic residues in domains A and B that are known to stack with the ssDNA bases are conserved in domain C, and as in domain A, one of these is required for viability in yeast. Interestingly, the amino-terminal portion of domain C contains a putative Cys4-type zinc-binding motif similar to that of another prokaryotic SSB, T4 gp32. We demonstrate that the ssDNA binding activity of domain C is uniquely sensitive to cysteine modification but that, as with gp32, ssDNA binding is not strictly dependent on zinc. The RPA heterotrimer is thus composed of at least four ssDNA binding domains and exhibits features of both bacterial and phage SSBs.

scholarly journals The iron–sulfur helicase DDX11 promotes the generation of single-stranded DNA for CHK1 activation

2020 ◽  
Vol 3 (3) ◽  
pp. e201900547 ◽  
Author(s):  
Anna K Simon ◽  
Sandra Kummer ◽  
Sebastian Wild ◽  
Aleksandra Lezaja ◽  
Federico Teloni ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Protein A ◽  
Dna Helicase ◽  
Binding Motif ◽  
Dependent Manner ◽  
Dna Polymerase Delta ◽  
Single Stranded Dna ◽  
Iron Sulfur ◽  
Replication Factors

The iron–sulfur (FeS) cluster helicase DDX11 is associated with a human disorder termed Warsaw Breakage Syndrome. Interestingly, one disease-associated mutation affects the highly conserved arginine-263 in the FeS cluster-binding motif. Here, we demonstrate that the FeS cluster in DDX11 is required for DNA binding, ATP hydrolysis, and DNA helicase activity, and that arginine-263 affects FeS cluster binding, most likely because of its positive charge. We further show that DDX11 interacts with the replication factors DNA polymerase delta and WDHD1. In vitro, DDX11 can remove DNA obstacles ahead of Pol δ in an ATPase- and FeS domain-dependent manner, and hence generate single-stranded DNA. Accordingly, depletion of DDX11 causes reduced levels of single-stranded DNA, a reduction of chromatin-bound replication protein A, and impaired CHK1 phosphorylation at serine-345. Taken together, we propose that DDX11 plays a role in dismantling secondary structures during DNA replication, thereby promoting CHK1 activation.

scholarly journals The GINS Complex from Pyrococcus furiosus Stimulates the MCM Helicase Activity

2007 ◽  
Vol 283 (3) ◽  
pp. 1601-1609 ◽  
Author(s):  
Takehiro Yoshimochi ◽  
Ryosuke Fujikane ◽  
Miyuki Kawanami ◽  
Fujihiko Matsunaga ◽  
Yoshizumi Ishino
Keyword(s):  
Dna Replication ◽  
Pyrococcus Furiosus ◽  
Dna Helicase ◽  
Hyperthermophilic Archaea ◽  
Eukaryotic Cells ◽  
Exponential Growth Phase ◽  
Chromosomal Dna ◽  
Mcm Helicase ◽  
Related Proteins

Pyrococcus furiosus, a hyperthermophilic Archaea, has homologs of the eukaryotic MCM (mini-chromosome maintenance) helicase and GINS complex. The MCM and GINS proteins are both essential factors to initiate DNA replication in eukaryotic cells. Many biochemical characterizations of the replication-related proteins have been reported, but it has not been proved that the homologs of each protein are also essential for replication in archaeal cells. Here, we demonstrated that the P. furiosus GINS complex interacts with P. furiosus MCM. A chromatin immunoprecipitation assay revealed that the GINS complex is detected preferentially at the oriC region on Pyrococcus chromosomal DNA during the exponential growth phase but not in the stationary phase. Furthermore, the GINS complex stimulates both the ATPase and DNA helicase activities of MCM in vitro. These results strongly suggest that the archaeal GINS is involved in both the initiation and elongation processes of DNA replication in P. furiosus, as observed in eukaryotic cells.

scholarly journals The WYL domain of the PIF1 helicase from the thermophilic bacteriumThermotoga elfiiis an accessory single-stranded DNA binding module

2017 ◽  
Author(s):  
Nicholas M. Andis ◽  
Christopher W. Sausen ◽  
Ashna Alladin ◽  
Matthew L. Bochman
Keyword(s):  
Unknown Function ◽  
Genome Stability ◽  
Dna Helicase ◽  
Thermophilic Bacterium ◽  
Helicase Domain ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Pif1 Helicase

ABSTRACTPIF1 family helicases are conserved from bacteria to man. With the exception of the well-studied yeast PIF1 helicases (e.g., ScPif1 and ScRrm3), however, very little is known about how these enzymes help maintain genome stability. Indeed, we lack a basic understanding of the protein domains found N- and C-terminal to the characteristic central PIF1 helicase domain in these proteins. Here, using chimeric constructs, we show that the ScPif1 and ScRrm3 helicase domains are interchangeable and that the N-terminus of ScRrm3 is important for its functionin vivo. This suggests that PIF1 family helicases evolved functional modules fused to a generic motor domain. To investigate this hypothesis, we characterized the biochemical activities of the PIF1 helicase from the thermophilic bacteriumThermotoga elfii(TePif1), which contains a C-terminal WYL domain of unknown function. Like helicases from other thermophiles, recombinant TePif1 was easily prepared, thermostablein vitro, and displayed activities similar to its eukaryotic homologs. We also found that the WYL domain was necessary for high-affinity single-stranded DNA (ssDNA) binding and affected both ATPase and helicase activities. Deleting the WYL domain from TePif1 or mutating conserved residues in the predicted ssDNA binding site uncoupled ATPase activity and DNA unwinding, leading to higher rates of ATP hydrolysis but less efficient DNA helicase activity. Our findings suggest that the domains of unknown function found in eukaryotic PIF1 helicases may also confer functional specificity and additional activities to these enzymes, which should be investigated in future work.

scholarly journals Differences in the Single-stranded DNA Binding Activities of MCM2-7 and MCM467

2007 ◽  
Vol 282 (46) ◽  
pp. 33795-33804 ◽  
Author(s):  
Matthew L. Bochman ◽  
Anthony Schwacha
Keyword(s):  
Dna Binding ◽  
Active Site ◽  
Biochemical Properties ◽  
Dna Helicase ◽  
Binding Activity ◽  
Helicase Activity ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Better Than

The MCM2-7 complex, a hexamer containing six distinct and essential subunits, is postulated to be the eukaryotic replicative DNA helicase. Although all six subunits function at the replication fork, only a specific subcomplex consisting of the MCM4, 6, and 7 subunits (MCM467) and not the MCM2-7 complex exhibits DNA helicase activity in vitro. To understand why MCM2-7 lacks helicase activity and to address the possible function of the MCM2, 3, and 5 subunits, we have compared the biochemical properties of the Saccharomyces cerevisiae MCM2-7 and MCM467 complexes. We demonstrate that both complexes are toroidal and possess a similar ATP-dependent single-stranded DNA (ssDNA) binding activity, indicating that the lack of helicase activity by MCM2-7 is not due to ineffective ssDNA binding. We identify two important differences between them. MCM467 binds dsDNA better than MCM2-7. In addition, we find that the rate of MCM2-7/ssDNA association is slow compared with MCM467; the association rate can be dramatically increased either by preincubation with ATP or by inclusion of mutations that ablate the MCM2/5 active site. We propose that the DNA binding differences between MCM2-7 and MCM467 correspond to a conformational change at the MCM2/5 active site with putative regulatory significance.

scholarly journals Single-Stranded DNA Binding by F TraI Relaxase and Helicase Domains Is Coordinately Regulated

2010 ◽  
Vol 192 (14) ◽  
pp. 3620-3628 ◽  
Author(s):  
Lubomír Dostál ◽  
Joel F. Schildbach
Keyword(s):  
Amino Acids ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Dna Helicase ◽  
Regulatory Function ◽  
Negative Cooperativity ◽  
Sequence Specificity ◽  
Wild Type ◽  
Single Stranded Dna ◽  
Ssdna Binding

ABSTRACT Transfer of conjugative plasmids requires relaxases, proteins that cleave one plasmid strand sequence specifically. The F plasmid relaxase TraI (1,756 amino acids) is also a highly processive DNA helicase. The TraI relaxase activity is located within the N-terminal ∼300 amino acids, while helicase motifs are located in the region comprising positions 990 to 1450. For efficient F transfer, the two activities must be physically linked. The two TraI activities are likely used in different stages of transfer; how the protein regulates the transition between activities is unknown. We examined TraI helicase single-stranded DNA (ssDNA) recognition to complement previous explorations of relaxase ssDNA binding. Here, we show that TraI helicase-associated ssDNA binding is independent of and located N-terminal to all helicase motifs. The helicase-associated site binds ssDNA oligonucleotides with nM-range equilibrium dissociation constants and some sequence specificity. Significantly, we observe an apparent strong negative cooperativity in ssDNA binding between relaxase and helicase-associated sites. We examined three TraI variants having 31-amino-acid insertions in or near the helicase-associated ssDNA binding site. B. A. Traxler and colleagues (J. Bacteriol. 188:6346-6353) showed that under certain conditions, these variants are released from a form of negative regulation, allowing them to facilitate transfer more efficiently than wild-type TraI. We find that these variants display both moderately reduced affinity for ssDNA by their helicase-associated binding sites and a significant reduction in the apparent negative cooperativity of binding, relative to wild-type TraI. These results suggest that the apparent negative cooperativity of binding to the two ssDNA binding sites of TraI serves a major regulatory function in F transfer.

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