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. Pull-down assays and surface plasmon resonance studies revealed that Cdc45-bound RPA complexed with ssDNA in the 8-10 nucleotide binding mode, but dissociated when RPA covered a 30-mer. Real-time analysis of RPA-ssDNA binding demonstrated that Cdc45 catalytically loaded RPA onto ssDNA. This placement reaction required physical contacts of Cdc45 with the RPA70A subdomain. Our results imply that Cdc45 controlled stabilization of the 8-nt RPA binding mode, the subsequent RPA transition into 30-mer mode and facilitated an ordered binding to ssDNA. We propose that a Cdc45-mediated loading guarantees a seamless deposition of RPA on newly emerging ssDNA at the nascent replication fork.

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. hCdc45 bound long (113-mer or 80-mer) single-stranded DNA fragments with a higher affinity than shorter ones (34-mer). hCdc45 displayed a preference for 3' protruding strands and bound tightly to single-strand/double-strand DNA junctions, such as those presented by Y-shaped DNA, bubbles and displacement loops, all of which appear transiently during the initiation of DNA replication. Collectively, our findings suggest that hCdc45 not only binds to but also slides on DNA with a 3'-5' polarity and, thereby acts as a molecular 'wedge' to initiate DNA strand displacement.

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. Biophysical characterization of these fragments revealed that the Sld2 homologous RecQL4 N-terminal domain carries large intrinsically disordered regions. The N-terminal fragments were sufficient for the strong annealing activity of RecQL4. Moreover, this activity appeared to be the basis for an ATP-independent strand exchange activity. Both activities relied on multiple DNA-binding sites with affinities to single-stranded, double-stranded and Y-structured DNA. Finally, we found a remarkable affinity of the N-terminus for guanine quadruplex (G4) DNA, exceeding the affinities for other DNA structures by at least 60-fold. Together, these findings suggest that the DNA interactions mediated by the N-terminal region of human RecQL4 represent a central function at the replication fork. The presented data may also provide a mechanistic explanation for the role of elements with a G4-forming propensity identified in the vicinity of vertebrate origins of DNA replication.

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. This is accompanied by morphological defects, elevation of senescence-associated ß-galactosidase levels, and changes in gene expression closely resembling those encountered during replicative (telomere-dependent) senescence. Activation of the p53 signaling pathway was found to be essential to this process. ChIP analysis and investigation of nascent DNA levels revealed that DHX9 is associated with origins of replication and that its suppression leads to a reduction of DNA replication. Our results demonstrate an essential role of DHX9 in DNA replication and normal cell cycle progression.

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. Several drug-sensitive shRNAs were identified that were selectively depleted in the presence of ABT-737. Of these, 2 independent shRNAs targeting the RNA/DNA helicase Dhx9 were found to sensitize lymphomas to ABT-737 to an extent comparable to control Mcl-1 shRNAs. Although Dhx9 suppression sensitized both mouse and human cells to ABT-737 treatment, it did so without altering MCL-1 levels. Rather, loss of Dhx9 appeared to activate a p53-dependent apoptotic program, through aggravation of replicative stress, which was found to be both necessary and sufficient for the ABT-737-shDhx9 synthetic lethal relationship.

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. We have identified the first 54 amino acids of RecQL4 (RecQL4_N54) as the minimum interaction region with human TopBP1. The solution structure of RecQL4_N54 was determined by heteronuclear liquid-state nuclear magnetic resonance (NMR) spectroscopy (PDB 2KMU; backbone root-mean-square deviation 0.73 Å). Despite low-sequence homology, the well-defined structure carries an overall helical fold similar to homeodomain DNA-binding proteins but lacks their archetypical, minor groove-binding N-terminal extension. Sequence comparison indicates that this N-terminal homeodomain-like fold is a common hallmark of metazoan RecQL4 and yeast Sld2 DNA replication initiation factors. RecQL4_N54 binds DNA without noticeable sequence specificity yet with apparent preference for branched over double-stranded (ds) or single-stranded (ss) DNA. NMR chemical shift perturbation observed upon titration with Y-shaped, ssDNA and dsDNA shows a major contribution of helix α3 to DNA binding, and additional arginine side chain interactions for the ss and Y-shaped DNA.

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. As TopBP1 binding partners we identified p54(nrb) and PSF, and confirmed the physical interactions by GST pull-down assays, co-immunoprecipitations and by yeast two-hybrid experiments. Recent evidence shows an involvement of p54(nrb) and PSF in DNA double-strand break repair (DSB) and radioresistance. To get a first picture of the physiological significance of the interaction of TopBP1 with p54(nrb) and PSF we investigated in real time the spatiotemporal behaviour of the three proteins after laser microirradiation of living cells. Localisation of TopBP1 at damage sites was noticed as early as 5 s following damage induction, whereas p54(nrb) and PSF localised there after 20 s. Both p54(nrb) and PSF disappeared after 20 s while TopBP1 was retained at damage sites significantly longer suggesting different functions of the proteins during DSB recognition and repair.

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. In contrast, DHX9 preferably unwinds RNA-RNA and RNA-DNA substrates, but fails to unwind Okazaki fragment-like hybrids. We further show that the preferential unwinding of RNA-containing substrates by WRN is stimulated by DHX9 in vitro, both on Okazaki fragment-like hybrids and on RNA-containing 'chicken-foot' structures. Collectively, our results suggest that WRN and DHX9 may also cooperate in vivo, e.g. at ongoing and stalled replication forks. In the latter case, the cooperation between both helicases may serve to form and to dissolve Holliday junction-like intermediates of regressed replication forks.

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. This complex mediates the loading of Cdc45. A second complex called GINS and consisting of Sld5 and Psf1, 2 and 3 is also loaded onto the origin during the initiation process, in an interdependent manner with the Sld2/Sld3/Dpb11 complex. Both complexes cooperate in the recruitment of the replicative DNA polymerases, thus executing the initiation and subsequent establishment of the replication fork. Cdc45 and GINS are essential, well-conserved factors that are retained at the elongating replication fork. They form a stable helicase complex with MCM2-7 and mediate its contact to the replicative DNA polymerases. In contrast, the Sld2/Sld3/Dpb11 complex critical for the initiation is not retained by the elongating replication fork. Sld2 displays limited homology to the amino-terminal region of RecQL4 helicase, which may represent its metazoan orthologue, whereas Sld3 homologues have been identified only in fungi. Dbp11 and its fission yeast homologue Cut5 are members of a large family of BRCT-containing proteins including human TopBP1 and fruit fly Mus101. Similar principles of regulation apply also to human initiation of DNA replication, despite obvious differences in the detailed mechanisms. The regulatory initiation cascade is intimately intertwined with the cell cycle apparatus as well as the checkpoint control.

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., H-DNA). Such structure-forming sequences are abundant and can regulate the expression of several disease-linked genes. In addition, the use of triplex-forming oligonucleotides (TFOs) to modulate gene structure and function has potential as an approach to targeted gene therapy. Previously, we found that endogenous H-DNA structures can induce DNA double-strand breaks and promote genomic rearrangements. Herein, we find that the DHX9 helicase co-immunoprecipitates with triplex DNA structures in mammalian cells, suggesting a role in the maintenance of genome stability. We tested this postulate by assessing the helicase activity of purified human DHX9 on various duplex and triplex DNA substrates in vitro. DHX9 displaced the third strand from a specific triplex DNA structure and catalyzed the unwinding with a 3' --> 5' polarity with respect to the displaced third strand. Helicase activity required a 3'-single-stranded overhang on the third strand and was dependent on ATP hydrolysis. The reaction kinetics consisted of a pre-steady-state burst phase followed by a linear, steady-state pseudo-zero-order reaction. In contrast, very little if any helicase activity was detected on blunt triplexes, triplexes with 5'-overhangs, blunt duplexes, duplexes with overhangs, or forked duplex substrates. Thus, triplex structures containing a 3'-overhang represent preferred substrates for DHX9, where it removes the strand with Hoogsteen hydrogen-bonded bases. Our results suggest the involvement of DHX9 in maintaining genome integrity by unwinding mutagenic triplex DNA structures.

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. Due to limited numbers of individuals screened to date little is known about the extent of these heterogeneities and how they are finally manifested in the proteins. Using expanded sample sizes, we show that sequence variations expressed as deletions or insertions of tri-nucleotides lead to different sized and structured repetitive units throughout a silk protein. Moreover, major ampullate spidroins 1 can quite dramatically differ in their overall lengths; however, extreme variants do not spread widely in a spider population. This suggests that a certain size range stabilized by purifying selection is important for spidroin 1 gene integrity and protein function. More than one locus for spidroin 1 genes possibly exist within one individual genome, which are homogenized in size, are differentially expressed and give a spider a certain degree of adaptation on silk's composition and properties. Such mechanisms are shared to a lesser extent by the second major ampullate spidroin gene.

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. The assay is based on a hairpin-forming biotinylated oligonucleotide substrate that contains one or more exonuclease-resistant phosphorothioate nucleotides. The activity and specificity of the selected 3'-5' exonuclease is determined indirectly using a sensitive pyrosequencing reaction after cleanup of the samples. In this pyrosequencing step, the amount of nucleotides filled into each position of the exonucleolytically degraded 3' end of the substrate can be recorded quantitatively and equals the amount of the nucleotides removed by the exonuclease. This system allows the estimation of both processivity and efficiency of the exonuclease activity. We have employed compounds reported in the literature to inhibit the exonuclease activities of either exonuclease III or the large fragment of polymerase I (Klenow fragment) to evaluate the assay.

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). To improve our understanding of the physiological functions of human TopBP1, we investigated the interplay between human TopBP1 and Cdc45 proteins in synchronized HeLa-S3 cells. Using GST (glutathione transferase) pull-down and co-immunoprecipitation techniques, we showed a direct interaction between TopBP1 and Cdc45 in vitro and in vivo. The use of deletion mutants in GST pull-down assays identified the first and second as well as the sixth BRCT domains of TopBP1 to be responsible for the functional interaction with Cdc45. Moreover, the interaction between Cdc45 and the first and second BRCT domains of TopBP1 inhibited their transcriptional activation both in yeast and mammalian one-hybrid systems. Both proteins interacted exclusively at the G(1)/S boundary of cell cycle; only weak interaction could be found at the G(2)/M boundary. The overexpression of the sixth BRCT domain led to diminished loading of Cdc45 on to chromatin. These results suggest that human TopBP1 is involved in the formation of the initiation complex of replication in human cells and is required for the recruitment of Cdc45 to origins of DNA replication.

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. Once the complex was engaged in the synthesis of an RNA primer and its extension, the interfering effects of the peptides ceased, suggesting a stable association of the replication factors during the initiation phase. Specific antibodies were still able to disrupt preformed interactions and inhibited primer synthesis and extension activities, underlining the crucial role of specific protein-protein contacts during the entire initiation process.

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. Inhibition by the peptide disrupting protein-protein interactions was observed only during the pre-initiation stage prior to primer synthesis, suggesting the formation of a stable initiation complex between RPA, Tag and Pol at the primer end.

The analysis of cell division and cell wall synthesis genes reveals mutationally inactivated ftsQ and mraY in a protoplast-type L-form of Escherichia coli.

Cell division and cell wall synthesis are tightly linked cellular processes for bacterial growth. A protoplast-type L-form Escherichia coli, strain LW1655F+, indicated that bacteria can divide without assembling a cell wall. However, the molecular basis of its phenotype remained unknown. To establish a first phenotype-genotype correlation, we analyzed its dcw locus, and other genes involved in division of E. coli. The analysis revealed defective ftsQ and mraY genes, truncated by a nonsense and a frame-shift mutation, respectively. Missense mutations were determined in the ftsA and ftsW products yielding amino-acid replacements at conserved positions. FtsQ and MraY, obviously nonfunctional in the L-form, are essential for cell division and cell wall synthesis, respectively, in all bacteria with a peptidoglycan-based cell wall. LW1655F+ is able to survive their loss-of-functions. This points to compensatory mechanisms for cell division in the absence of murein sacculus formation. Hence, this L-form represents an interesting model to investigate the plasticity of cell division in E. coli, and to demonstrate how concepts fundamental for bacterial life can be bypassed.

p53 stimulates human topoisomerase I activity by modulating its DNA binding.

The tumor suppressor protein p53 and the human DNA topoisomerase I (htopoI) interact with each other, which leads to a stimulation of the catalytic activity of htopoI. Moreover, p53 stimulates the topoisomerase I-induced recombination repair (TIRR) reaction. However, little was known about how p53 stimulates this topoisomerase I activity. Here we demonstrate that monomeric p53 is sufficient for the stimulation of the topoisomerase I-catalyzed relaxation activity, but the tetrameric form of p53 is required for the stimulation of TIRR. We also show that p53 stimulates topoisomerase I activity by increasing the dissociation of htopoI from DNA. Since htopoI forms a closed ring structure around the DNA, our results suggest that p53 induces a conformational change within htopoI that results in an opening of the clamp, and thereby releases htopoI from DNA.

Human topoisomerase I cleavage complexes are repaired by a p53-stimulated recombination-like reaction in vitro.

Several studies have shown that human topoisomerase I (htopoI) cleaves in the vicinity of various DNA lesions and thereby forms covalent intermediates known as 'cleavage complexes'. Such complexes are detrimental to cells if they are not repaired. Therefore, it is generally accepted that repair pathways must exist for such lesions. We have demonstrated that a htopoI cleavage complex can be recognized by a second topoisomerase I molecule and thereby perform a so-called htopoI 'double cleavage' in vitro. In addition, we found that the double cleavage reaction was stimulated by p53. Here we show that the double cleavage reaction results in the removal of the original htopoI cleavage complex and the generation of a single-stranded gap of approximately 13 nt. This gap supports a sequence-dependent DNA recombination reaction mediated by the second htopoI molecule. Furthermore, we show that p53 strongly stimulates the recombination reaction. We suggest that this reaction may represent a novel p53-dependent topoisomerase I-induced recombination repair (TIRR) pathway for htopoI cleavage complexes.

DNA-dependent protein kinase (DNA-PK) phosphorylates nuclear DNA helicase II/RNA helicase A and hnRNP proteins in an RNA-dependent manner.

An RNA-dependent association of Ku antigen with nuclear DNA helicase II (NDH II), alternatively named RNA helicase A (RHA), was found in nuclear extracts of HeLa cells by immunoprecipitation and by gel filtration chromatography. Both Ku antigen and NDH II were associated with hnRNP complexes. Two-dimensional gel electrophoresis showed that Ku antigen was most abundantly associated with hnRNP C, K, J, H and F, but apparently not with others, such as hnRNP A1. Unexpectedly, DNA-dependent protein kinase (DNA-PK), which comprises Ku antigen as the DNA binding subunit, phosphorylated hnRNP proteins in an RNA-dependent manner. DNA-PK also phosphorylated recombinant NDH II in the presence of RNA. RNA binding assays displayed a preference of DNA-PK for poly(rG), but not for poly(rA), poly(rC) or poly(rU). This RNA binding affinity of DNA-PK can be ascribed to its Ku86 subunit. Consistently, poly(rG) most strongly stimulated the DNA-PK-catalyzed phosphorylation of NDH II. RNA interference studies revealed that a suppressed expression of NDH II altered the nuclear distribution of hnRNP C, while silencing DNA-PK changed the subnuclear distribution of NDH II and hnRNP C. These results support the view that DNA-PK can also function as an RNA-dependent protein kinase to regulate some aspects of RNA metabolism, such as RNA processing and transport.

Initiation of eukaryotic DNA replication: regulation and mechanisms.

The accurate and timely duplication of the genome is a major task for eukaryotic cells. This process requires the cooperation of multiple factors to ensure the stability of the genetic information of each cell. Mutations, rearrangements, or loss of chromosomes can be detrimental to a single cell as well as to the whole organism, causing failures, disease, or death. Because of the size of eukaryotic genomes, chromosomal duplication is accomplished in a multiparallel process. In human somatic cells between 10,000 and 100,000 parallel synthesis sites are present. This raises fundamental problems for eukaryotic cells to coordinate the start of DNA replication at each origin and to prevent replication of already duplicated DNA regions. Since these general phenomena were recognized in the middle of the 20th century the regulation and mechanisms of the initiation of eukaryotic DNA replication have been intensively investigated. These studies were carried out to find the essential factors involved in the process and to determine their functions during DNA replication. These studies gave rise to a model of the organization and the coordination of DNA replication within the eukaryotic cell. The elegant experiments carried out by Rao and Johnson (1970) (1), who fused cells in different phases of the cell cycle, showed that G1 cells are competent for replication of their chromosomes, but lack a specific diffusible factor required to activate their replicaton machinery and showed that G2 cells are incompetent for DNA replication. These findings suggested that eukaryotic cells exist in two states. In G1 phase, cells are competent to initiate DNA replication, which is subsequently triggered in S phase. After completion of S phase, cells in G2 are no longer able to initiate DNA replication and they require a transition through mitosis to reenable initiation of DNA replication to take place in the next S phase. The Xenopus cell-free replication system has proved a good model system in which to study DNA replication in vitro as well as the mechanism preventing rereplication within a single cell cycle (2). Studies using this system resulted in the development of a model postulating the existence of a replication licensing factor, which binds to chromatin before the G1-S transition and which is displaced during replication (2, 3). These results were supported by genetic and biochemical experiments in Saccharomyces cerevisiae (budding yeast) and Schizosaccharomyces pombe (fission yeast) (4, 5). The investigation of cell division cycle mutants and the budding yeast origin of replication resulted in the concept of a prereplicative and a postreplicative complex of initiation proteins (6-9). These three individual concepts have recently started to merge and it has become obvious that initiation in eukaryotes is generally governed by the same ubiquitous mechanisms.