Cdc45-induced loading of human RPA onto single-stranded DNA.

Cell division cycle protein 45 (Cdc45) is an essential component of the eukaryotic replicative DNA helicase. We found that human Cdc45 forms a complex with the single-stranded DNA (ssDNA) binding protein RPA. Moreover, it actively loads RPA onto nascent ssDNA.

Human DNA polymerase α interacts with mismatch repair proteins MSH2 and MSH6.

High fidelity of genome duplication is ensured by cooperation of polymerase proofreading and mismatch repair (MMR) activities. Here, we show that human mismatch recognizing proteins MutS homolog 2 (MSH2) and MSH6 copurify and interact with replicative Pol α. This enzyme also is the replicative primase and replicates DNA with poor fidelity.

S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci.

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is an essential process in maintenance of chromosomal stability. A key player of HR is the strand exchange factor RAD51 whose assembly at sites of DNA damage is tightly regulated. We detected an endogenous complex of RAD51 with the calcium-binding protein S100A11, which is localized at sites of DNA repair in HaCaT cells as well as in normal human epidermal keratinocytes (NHEK) synchronized in S phase.

Cdc45 is limiting for replication initiation in humans.

Cdc45 is an essential protein that together with Mcm2-7 and GINS forms the eukaryotic replicative helicase CMG. Cdc45 seems to be rate limiting for the initial unwinding or firing of replication origins. In line with this view, Cdc45-overexpressing cells fired at least twice as many origins as control cells.

Time-Resolved In Situ Spectroscopy During Formation of the GaP/Si(100) Heterointerface.

Though III-V/Si(100) heterointerfaces are essential for future epitaxial high-performance devices, their atomic structure is an open historical question. Benchmarking of transient optical in situ spectroscopy during chemical vapor deposition to chemical analysis by X-ray photoelectron spectroscopy enables us to distinguish between formation of surfaces and of the heterointerface. A terrace-related optical anisotropy signal evolves during pulsed GaP nucleation on single-domain Si(100) surfaces.

The intrinsically disordered amino-terminal region of human RecQL4: multiple DNA-binding domains confer annealing, strand exchange and G4 DNA binding.

Human RecQL4 belongs to the ubiquitous RecQ helicase family. Its N-terminal region represents the only homologue of the essential DNA replication initiation factor Sld2 of Saccharomyces cerevisiae, and also participates in the vertebrate initiation of DNA replication. Here, we utilized a random screen to identify N-terminal fragments of human RecQL4 that could be stably expressed in and purified from Escherichia coli.

Suppression of the DHX9 helicase induces premature senescence in human diploid fibroblasts in a p53-dependent manner.

DHX9 is an ATP-dependent DEXH box helicase with a multitude of cellular functions. Its ability to unwind both DNA and RNA, as well as aberrant, noncanonical polynucleotide structures, has implicated it in transcriptional and translational regulation, DNA replication and repair, and maintenance of genome stability. We report that loss of DHX9 in primary human fibroblasts results in premature senescence, a state of irreversible growth arrest.

DNA binding properties of human Cdc45 suggest a function as molecular wedge for DNA unwinding.

The cell division cycle protein 45 (Cdc45) represents an essential replication factor that, together with the Mcm2-7 complex and the four subunits of GINS, forms the replicative DNA helicase in eukaryotes. Recombinant human Cdc45 (hCdc45) was structurally characterized and its DNA-binding properties were determined. Synchrotron radiation circular dichroism spectroscopy, dynamic light scattering, small-angle X-ray scattering and atomic force microscopy revealed that hCdc45 exists as an alpha-helical monomer and possesses a structure similar to its bacterial homolog RecJ.

RNAi screening uncovers Dhx9 as a modifier of ABT-737 resistance in an Eμ-myc/Bcl-2 mouse model.

ABT-737 is a promising chemotherapeutic agent that promotes apoptosis by acting as a selective BH3 mimetic to neutralize Bcl-2-like family members. One shortcoming with its use is that Mcl-1, a member of the Bcl-2 family, is poorly inhibited by ABT-737 and thus is a major cause of resistance. We performed a short hairpin RNA (shRNA)-based drop-out screen to identify novel genes and pathways that could reverse resistance to ABT-737 treatment in Eµ-myc/Bcl-2 lymphoma cells engineered to rely on endogenous Mcl-1 for survival.

The N-terminus of the human RecQL4 helicase is a homeodomain-like DNA interaction motif.

The RecQL4 helicase is involved in the maintenance of genome integrity and DNA replication. Mutations in the human RecQL4 gene cause the Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes. Mouse models and experiments in human and Xenopus have proven the N-terminal part of RecQL4 to be vital for cell growth.

Proteomic identification of PSF and p54(nrb) as TopBP1-interacting proteins.

TopBP1 is a BRCT domain-rich protein that is structurally and functionally conserved throughout eukaryotic organisms. It is required for the initiation of DNA replication and for DNA repair and damage signalling. To further dissect its biological functions, we explored TopBP1-interacting proteins by co-immunoprecipitation assays and LC-ESI-MS-analyses.

Human DHX9 helicase preferentially unwinds RNA-containing displacement loops (R-loops) and G-quadruplexes.

Human DHX9 helicase, also known as nuclear DNA helicase II (NDH II) and RNA helicase A (RHA), belongs to the SF2 superfamily of nucleic acid unwinding enzymes. DHX9 melts simple DNA-DNA, RNA-RNA, and DNA-RNA strands with a 3'-5' polarity; despite this little is known about its substrate specificity. Here, we used partial duplex DNA consisting of M13mp18 DNA and oligonucleotide-based replication and recombination intermediates.

Human DHX9 helicase unwinds triple-helical DNA structures.

Naturally occurring poly(purine.pyrimidine) rich regions in the human genome are prone to adopting non-canonical DNA structures such as intramolecular triplexes (i.e.

A label-free assay of exonuclease activity using a pyrosequencing technique.

Enzymes with 3'-5' exonuclease activities are important in promoting the accuracy of DNA replication and DNA repair by proofreading. The alteration of the function of these enzymes by endogenous or exogenous effectors could, therefore, have a considerable impact on DNA replication and ultimately on genome integrity. We have developed a label-free high-throughput screening method for quantifying the effects of different reagents on exonuclease activity.

WRN helicase unwinds Okazaki fragment-like hybrids in a reaction stimulated by the human DHX9 helicase.

Mutations in the Werner gene promote the segmental progeroid Werner syndrome (WS) with increased genomic instability and cancer. The Werner gene encodes a DNA helicase (WRN) that can engage in direct protein-protein interactions with DHX9, also known as RNA helicase A or nuclear DNA helicase II, which represents an essential enzyme involved in transcription and DNA repair. By using several synthetic nucleic acid substrates we demonstrate that WRN preferably unwinds RNA-containing Okazaki fragment-like substrates suggesting a role in lagging strand maturation of DNA replication.

Containment of extended length polymorphisms in silk proteins.

The spider silk gene family to the current date has been developed by gene duplication and homogenization events as well as conservation of crucial sequence parts. These evolutionary processes have created an amazing diversity of silk types each associated with specific properties and functions. In addition, they have led to allelic and gene variants within a species as exemplified by the major ampullate spidroin 1 gene of Nephila clavipes.

Molecular characterization of nuclear DNA helicase II (RNA helicase A).

Nuclear DNA helicase II (NDH II) was first isolated from calf thymus using a DNA-unwinding assay. Subsequently it has been shown to be a homologue of human RNA helicase A (RHA) and the maleless protein (MLE) from Drosophila. Accordingly, the protein possesses both DNA and RNA unwinding activities.

The initiation step of eukaryotic DNA replication.

Eukaryotic initiation of DNA replication is a tightly regulated process. In the yeasts, S-phase-specific cyclin Cdk1 complex as well as Dfb4-Cdc7 kinase phosphorylate the initiation factors Sld2 and Sld3. These factors form a ternary complex with another initiation factor Dbp11 in their phosphorylated state, and associate with the origin of replication.

Composition and hierarchical organisation of a spider silk.

Albeit silks are fairly well understood on a molecular level, their hierarchical organisation and the full complexity of constituents in the spun fibre remain poorly defined. Here we link morphological defined structural elements in dragline silk of Nephila clavipes to their biochemical composition and physicochemical properties. Five layers of different make-ups could be distinguished.

Characterization of the interaction between the human DNA topoisomerase IIbeta-binding protein 1 (TopBP1) and the cell division cycle 45 (Cdc45) protein.

TopBP1 (topoisomerase IIbeta-binding protein 1) is a BRCT [BRCA1 (breast-cancer susceptibility gene 1) C-terminal]-domain-rich protein that is structurally and functionally conserved throughout eukaryotic organisms. It is required for the initiation of DNA replication and for DNA repair and DNA damage signalling. Experiments with fission yeast and Xenopus revealed that the TopBP1 homologues of these organisms are required for chromatin loading of the replication protein Cdc45 (cell division cycle 45).

Different activities of the largest subunit of replication protein A cooperate during SV40 DNA replication.

Replication protein A (RPA) is a stable heterotrimeric complex consisting of p70, p32 and p14 subunits. The protein plays a crucial role in SV40 minichromosome replication. Peptides of p70 representing interaction sites for the smaller two subunits, DNA as well as the viral initiator protein large T-antigen (Tag) and the cellular DNA polymerase alpha-primase (Pol) all interfered with the replication process indicating the importance of the different p70 activities in this process.

Timed interactions between viral and cellular replication factors during the initiation of SV40 in vitro DNA replication.

The initiation of SV40 (simian virus 40) DNA replication requires the co-operative interactions between the viral Tag (large T-antigen), RPA (replication protein A) and Pol (DNA polymerase alpha-primase) on the template DNA. Binding interfaces mapped on these enzymes and expressed as peptides competed with the mutual interactions of the native proteins. Prevention of the genuine interactions was accomplished only prior to the primer synthesis step and blocked the assembly of a productive initiation complex.

Interactions of human Cdc45 with the Mcm2-7 complex, the GINS complex, and DNA polymerases delta and epsilon during S phase.

Cdc45 is an essential cellular protein that functions in both the initiation and elongation of DNA replication. Here, we analyzed the localization of human Cdc45 and its interactions with other proteins during the cell cycle. Human Cdc45 showed a diffuse distribution in G1 phase, a spot-like pattern in S and G2, and again a diffuse distribution in M phase of the cell cycle.