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PubMed1988

The primosomal protein n' of Escherichia coli is a DNA helicase.

Protein n' of Escherichia coli functions in assembly and translocation of the primosome, a mobile multiprotein complex involved in priming DNA replication (Kornberg, A. (1982) Supplement to DNA Replication, Freeman Publications, San Francisco). By itself, protein n' translocates on single-stranded DNA and destabilizes duplex regions by acting as a DNA helicase, using the energy of ATP or dATP hydrolysis. Single-stranded DNA binding protein was required for melting of duplex regions longer than 40 base pairs. Initial binding of protein n' to a specific site on DNA (Shlomai, J., and Kornberg, A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 799-803) is essential for its helicase function. The polarity of protein n' translocation on DNA, in the 3' to 5' direction of the chain, suggests a mechanism for how the primosome may contribute to concurrent replication of both strands at a replication fork.

Adenosine Triphosphate↗

PubMed1987

The regulatory protein GAL80 is a determinant of the chromatin structure of the yeast GAL1-10 control region.

Chromatin in the regions between the upstream activator sequence and the 5' ends of the yeast GAL1 and GAL10 genes has been analyzed by DNase I chromosomal footprinting and micrococcal nuclease digestion using the indirect end-labeling approach. Comparison of wild type chromatin digests to naked DNA digests shows that there are specific regions of these upstream sequences which are strongly protected in chromatin. Comparison to chromatin digests from cells disrupted for the positive regulatory gene, GAL4, or the negative regulatory gene, GAL80, and thus lacking GAL4 or GAL80 function, shows that these regions of protection in wild type chromatin are GAL80-dependent but not GAL4-dependent. The protected regions include DNA lying on (GAL10) or near (GAL1) the respective TATA boxes. These protections are present in both noninduced and induced cells. Both DNA strands are equally protected. Upstream of GAL1 there is a second protected region. This protection shows considerable expression and strand dependence. These observations provide the first evidence that the GAL80 function influences chromatin structure and suggest possible mechanisms by which GAL80 modulates the GAL1 and 10 promoters in induced cells. Micrococcal nuclease digests also suggest a role for GAL80 in a distinctive higher order organization of the intergenic region, perhaps involving multiprotein complexes.

Chromatin↗

PubMed1989

Phosphorylation of HeLa cell multiprotein DNA polymerase alpha complex: impact on activity and partial purification of the associated kinase.

Phosphorylation is a major post-translational regulatory mechanism and plays a key role in transduction of mitogenic signals in cell proliferation. The role of phosphorylation and dephosphorylation in regulating the activities of a multiprotein DNA polymerase alpha complex was examined. Treatment of the HeLa cell multiprotein DNA polymerase alpha with calf intestinal alkaline phosphatase resulted in the inactivation of DNA polymerase alpha and DNA primase but had no effect on deoxyribonuclease- and primer-recognition proteins. A protein kinase co-purified with the multiprotein DNA polymerase alpha and was partially purified from HeLa cells. The partially purified kinase was active in phosphorylating dephosphorylated polymerase alpha and used casein and histones as exogenous substrates. This study demonstrates that phosphorylation-dephosphorylation may have modulated the activities of DNA replicative enzymes and suggests a role for specific phosphatases and kinases in this process.

DNA↗

PubMed1986

Selection of template initiation sites and the lengths of RNA primers synthesized by DNA primase are strongly affected by its organization in a multiprotein DNA polymerase alpha complex.

Synthesis of (p)ppRNA-DNA chains by purified HeLa cell DNA primase-DNA polymerase alpha (pol alpha-primase) was compared with those synthesized by a multiprotein form of DNA polymerase alpha (pol alpha 2) using unique single-stranded DNA templates containing the origin of replication for simian virus 40 (SV40) DNA. The nucleotide locations of 33 initiation sites were identified by mapping G*pppN-RNA-DNA chains and identifying their 5'-terminal ribonucleotide. Pol alpha 2 strongly preferred initiation sites that began with ATP rather than GTP, thus frequently preferring different initiation sites than pol alpha-primase, depending on the template examined. The initiation sites selected in vitro, however, did not correspond to the sites used during SV40 DNA replication in vivo. Pol alpha 2 had the greatest effect on RNA primer size, typically synthesizing primers 1-5 nucleotides long, while pol alpha-primase synthesized primers 6-8 nucleotides long. These differences were observed even at individual initiation sites. Thus, the multiprotein form of DNA primase-DNA polymerase alpha affects selection of initiation sites, the frequency at which the sites are chosen, and length of RNA primers.

Base Sequence↗

PubMed1987

Participation of ATP in the binding of a yeast replicative complex to DNA.

The activity that replicates yeast DNA in vitro can be isolated from cells of the budding yeast Saccharomyces in a high-Mr (approximately 2 X 10(6] form. Several lines of evidence indicate that this fraction contains a multiprotein replicative complex. A functional assay has been developed for the analysis of the interaction of the replicating activity with DNA. Binding of the activity required Mg2+, but did not require the addition of ATP or the other ribo- or deoxynucleoside triphosphates. However, the ATP analogues adenosine 5'-[gamma-thio]triphosphate and adenosine 5'-[beta gamma-imido]triphosphate blocked the binding, suggesting that ATP participates in the interaction at some stage. The binding was template (origin)-specific in either the presence or the absence of ATP and the other nucleoside triphosphates; however, ATP stabilized the replicating activity. The preferential inhibition of binding that was observed in the presence of the DNA topoisomerase II inhibitor coumermycin suggests that the requirement for ATP may be at least partially accounted for by the involvement of this enzyme in the initial interaction of the replicating activity with DNA. Finally, the binding was rapid. In contrast, DNA synthesis displayed a lag when assayed directly without first allowing a period for the replicating activity to bind to the DNA. In addition, binding was 'tight', as judged by the resistance of the protein--DNA complexes to salt in comparison with the relative sensitivity of binding. The replicating activity was not readily displaced from the complexes by exogenous DNAs, either possessing or lacking yeast origins of replication. The results suggest that the interaction of the replicating activity with the DNA occurs in more than one stage.

Adenosine Triphosphate↗

PubMed1991

Purification and interaction properties of the human RNA polymerase B(II) general transcription factor BTF2.

A general transcription factor (BTF2) has been purified from HeLa whole cell extract and shown to be absolutely required for the formation of a functional initiation complex. We also demonstrate that this factor binds in solution to RNA polymerase B(II) and to the other general transcription factors BTF1(TFIID), BTF3, and BTF4. These results strongly suggest the existence of multiple interactions between the various components of the multiprotein initiation complex.

HeLa Cells↗

PubMed1986

Genetic aspects of cellular interactions in the immune response.

Our understanding of the complex cellular interactions responsible for mediating effective immune responses has increased substantially in recent years. It is now clear that the genetic loci that control the interaction of the cells of the immune response encode groups of closely related cell-surface molecules. These molecules are the class I and class II antigens of the MHC, the differentiation antigens on lymphocyte subpopulations, and the receptors of various types, including the membrane immunoglobulin of B lymphocytes and the antigen receptors of T lymphocytes. Biochemical analysis of these cell surface molecules has demonstrated that they display important DNA sequence homologies. A polypeptide of approximately 110 amino acids comprises the basic building block for many of the cell surface molecules. Gradually, as a consequence of evolutionary development, the immune system has expanded its ability to respond to the external environment by an increased complexity of lymphocyte subpopulations and the surface structures that modulate their interaction. These cell surface molecules provide the structures that allow collaborative interaction of different cell types and that form the multiprotein receptor complexes involved in the recognition of, and specific response to, foreign antigens. Our future understanding of the control of the immune response will depend upon establishing the biochemical nature and the multifaceted interactions of these important molecules.

Animals↗

PubMed1987

Evidence for the involvement of a single major species of replicative complex in DNA synthesis from two diverse nuclear replicons in yeast.

The activity that replicates the 2-micron yeast DNA plasmid in vitro can be isolated as a high-molecular weight (approximately 2 X 10(6)) fraction, which possesses many of the features of a multiprotein replicative complex. This fraction also initiates DNA synthesis at the yeast chromosomal replicator ARS1 raising the question whether the preparations discriminate between origins. It was determined that the binding of replicative complex to plasmids containing either 2-microns or ARS1 origins of replication was indistinguishable. The preparations also showed no preference among them for replication. In addition, the DNAs competed with each other to the same extent for binding of replicative complex. These results suggest that these two origins share one major species of replicative complex.

Binding, Competitive↗

PubMed2026

Maternal redd1 mRNA decline triggers mTORC1 activation during the blastula-gastrula transition in zebrafish embryos.

During early metazoan development, maternal mRNAs and proteins stored in the egg sustain initial cellular functions. After the blastula stage, developmental control shifts to zygotic gene expression, and maternal transcripts are progressively degraded. Although mTORC1 is a central regulator of global mRNA translation and cell growth, its role in controlling maternal mRNA translation prior to gastrulation remains poorly understood. In zebrafish embryos, the mTORC1 inhibitor redd1 is abundantly expressed after fertilization but decreases following the maternal-to-zygotic transition (MZT), inversely correlating with mTORC1 activity. Overexpression of redd1 suppresses mTORC1, impairs gastrulation, and reduces translation of 5'TOP mRNAs and key regulatory genes, underscoring the necessity of relieving mTORC1 inhibition after the blastula stage. To investigate redd1 translation under conditions of low mTORC1 activity, we injected reporter mRNAs containing its 5' and 3' UTRs. The 3'UTR promoted polyadenylation and enhanced translation, while both UTRs enabled efficient reporter expression despite mTORC1 suppression, indicating that redd1 mRNA is translated independently of canonical mTORC1 pathways. Similarly, maternal mRNAs such as nanog, myca, pou5f3, and ccnb1, as well as the early zygotic transcript dharma, are translated through mTORC1-independent mechanisms. Together, these findings reveal a transient phase of mTORC1 suppression in early zebrafish embryos and demonstrate that select maternal and zygotic mRNAs bypass this regulation to ensure proper developmental progression.

Animals↗

PubMed1987

Electron microscopy of nickel-containing methanogenic enzymes: methyl reductase and F420-reducing hydrogenase.

Methanogens catalyze the hydrogen-dependent eight-electron reduction of carbon dioxide to methane. Two of the key catalysts in the eight-electron reduction pathway are the nickel-containing enzymes F420-reducing hydrogenase and methyl reductase. In the present study, the structures of these archaebacterial enzymes from Methanobacterium thermoautotrophicum delta H have been determined by electron microscopy. By negative stain techniques, F420 hydrogenase was found to be a ring structure with a diameter of 15.7 nm and an inner channel 4 nm in diameter. Shadow-casting experiments demonstrated that the rings were 8.5 nm deep, indicating a holoenzyme molecular weight of 8.0 X 10(5). Methyl reductase appeared to be an oligomeric complex of dimensions 8.5 by 9 by 11 nm, with a central stain-penetrating region. The morphology and known subunit composition suggest a model in which the subunits are arranged as an eclipsed pair of open trimers. Methyl reductase was also found in the form of larger aggregates and in paracrystalline arrays derived from highly concentrated solutions. The extremely large size of F420 hydrogenase and the methyl reductase supramolecular assemblies may have relevance in vivo in the construction of multiprotein arrays that function in methane biogenesis.

Euryarchaeota↗

PubMed2026

Condensin accelerates long-range intra-chromosomal interactions.

The 3D genome organization plays a key role in regulating interactions among chromosomal loci. While Chromosome Conformation Capture (3C)-based methods have provided static snapshots of chromatin architecture, the kinetics of chromosomal encounters in live cells remain poorly characterized. In this study, we employ Chemically Induced Chromosomal Interaction (CICI) to measure encounter times between multiple loci pairs in G1-arrested budding yeast. Our results show that chromosome motion closely follows the Rouse polymer model, with similar diffusion parameters at all tested loci. Surprisingly, we find that long-range intra-chromosomal encounters occur significantly faster than inter-chromosomal encounters at similar 3D distances. Using targeted depletion experiments, we identify condensin, but not cohesin, as the complex mostly responsible for these rapid intra-chromosomal interactions. This is further supported by Hi-C analysis, which reveals that condensin promotes long-distance intra-chromosomal interactions in G1 yeast. Through polymer simulations, we estimate that condensin extrudes chromatin at ~2 kb/s with a density of one complex per 1-2 Mb and a processivity of 120-220 kb. These findings uncover a novel role for condensin in shaping the interphase genome organization and provide new insights into chromosomal search dynamics in vivo.

Saccharomyces cerevisiae↗

PubMed2026

Bridging-driven condensation by eukaryotic SMC complexes is a conserved feature of genome organization.

The Structural Maintenance of Chromosome (SMC) protein family plays a central role in higher-order genome organization through ATP-dependent DNA loop extrusion by cohesin and condensin and other processes. Whether these activities fully account for the complexity of chromosome architecture remains unknown. Here, we uncover a conserved ATP-independent mechanism of chromatin condensation by SMC complexes, occurring via biomolecular condensation. Using single-molecule fluorescence imaging, we show that a variety of SMCs form dynamic DNA-bound condensates that exhibit key features of biomolecular condensates, including droplet coalescence, fluorescence recovery after photobleaching, and rapid exchange with free SMC complexes. Atomic force microscopy analysis of human cohesin-DNA assemblies reveals DNA-length-dependent clustering, providing evidence for bridging-driven condensation. Analyses of in vivo super-resolution imaging and high-throughput chromosome conformation capture (Hi-C) data indicate that these condensates form chromatin-associated clusters with multi-loop structures. Together, our results establish that SMC complexes employ ATP-independent phase condensation as well as ATP-dependent activities to shape genome architecture. This work reveals a broadly conserved principle of chromosomal organization across eukaryotes.

Chromosomal Proteins, Non-Histone↗

PubMed2025

WDFY2 promotes MRN complex formation required for homologous recombination-mediated DNA repair.

The MRE11-RAD50-NBS1 (MRN) complex is fundamental for detecting and repairing DNA double-strand breaks (DSBs), thereby safeguarding genome integrity. However, the precise mechanism governing MRN complex recruitment to DSBs remains largely unexplored. Here, we identify WD40- and FYVE domain-containing protein 2 (WDFY2) as an important regulator of MRN complex formation at DNA damage sites, facilitating homologous recombination (HR) repair. Mechanistically, WDFY2 is phosphorylated at serine 84 by the ATM-CHK2 axis, priming it for recruitment to DSBs. Through direct interactions with MRE11 and NBS1, WDFY2 bridges the MRE11-RAD50 subcomplex with NBS1, thereby promoting MRN complex formation at DSBs and DNA end resection. WDFY2 deficiency, as well as the non-phosphorylatable S84A mutant, results in impaired HR repair and reduced cell survival following DNA damage. Collectively, our findings establish WDFY2 as a key platform for MRN complex loading at DSBs and HR repair, highlighting it as a potential therapeutic target for cancer treatment.

Humans↗

PubMed2026

BCDX2-CX3 and DX2-CX3 complexes assemble and stabilize RAD51 filaments.

The repair of DNA double-strand breaks by homologous recombination is essential for genomic integrity, and its dysregulation is a hallmark of cancer1. Central to homologous recombination is the RAD51 recombinase, whose assembly into a nucleoprotein filament is governed by five RAD51 paralogues (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3)2. Mutations in any of these proteins predispose individuals to multiple cancers or genetic disorders3-6. These paralogues are thought to form two functionally separate complexes RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) and RAD51C-XRCC3 (CX3), that act independently at different stages of homologous recombination7-11. Here we demonstrate that all five paralogues can assemble into a single, ATP-dependent BCDX2-CX3-RAD51 supercomplex. The architecture of this assembly bound to single-stranded DNA reveals a contiguous filament where the CX3 module stacks atop BCDX2, creating a protofilament template for RAD51 filament formation. We further identify a novel, RAD51B-independent DX2-CX3 complex (RAD51D-XRCC2-RAD51C-XRCC3) functioning as a stable RAD51 anchor on single-stranded DNA, and we capture it in multiple states, including capping RAD51 filament segment. These distinct assemblies are differentially regulated by ATPase activity, defining a dynamic BCDX2-CX3 'loader' and a stable DX2-CX3 'anchor' that provide functional modularity to the homologous recombination machinery. This work provides a unifying mechanism for human RAD51 paralogue function and delivers an atomic blueprint for interpreting disease-causing mutations.

Rad51 Recombinase↗

PubMed2025

Zincore, an atypical coregulator, binds zinc finger transcription factors to control gene expression.

Zinc finger proteins (ZNFs) are the largest family of transcription factors, yet how they activate gene expression remains unclear. In this study, we identified Zincore, a protein complex consisting of QRICH1 and SEPHS1, as a ZNF-specific coregulator essential for embryonic development in mice and associated with developmental syndromes in humans. We also identified ZFP91 as a representative Zincore client, binding the conserved promoter motif CTTTAAR. Cryo-electron microscopy of a Zincore-ZFP91-DNA complex revealed a SEPHS1 arginine clamp to recognize the DNA-bound zinc finger domains. This mode of binding explains recognition of different ZNFs and stabilizes ZFP91 onto its cognate DNA motif. Thus, our study identified Zincore as a ZNF-specific coregulator essential for development, involving a distinctive mechanism that locks ZNFs onto DNA and regulates transcription.

Animals↗

PubMed1991

Structural analysis by circular dichroism of some enzymes involved in plant cell wall degradation.

Three enzymes which degrade different polysaccharide components of plant cell walls have been characterized by circular dichroism (CD). A bacterial endoglucanase, which in the native state forms part of a multiprotein cellulase complex, showed a tendency to form aggregates as measured by CD. Depending on its degree of aggregation, this enzyme displayed between 50% and 100% helical structure, whereas a bacterial xylanase and a fungal polygalacturonase exhibited more beta-sheet structure. The polygalacturonase was apparently devoid of helical structure.

Cell Wall↗

PubMed2024

Alpha- and betacoronavirus cis-acting RNA elements.

Coronaviruses have exceptionally large RNA genomes and employ multiprotein replication/transcription complexes to orchestrate specific steps of viral RNA genome replication and expression. Most of these processes involve viral cis-acting RNA elements that are engaged in vital RNA-RNA and/or RNA-protein interactions. Over the past years, a large number of studies provided interesting new insight into the structures and, to a lesser extent, functions of specific RNA elements for representative coronaviruses, and there is evidence to suggest that (a majority of) these RNA elements are conserved across genetically divergent coronavirus genera. It is becoming increasingly clear that at least some of these elements do not function in isolation but operate through complex and highly dynamic RNA-RNA interactions. This article reviews structural and functional aspects of cis-acting RNA elements conserved in alpha- and betacoronavirus 5'- and 3'-terminal genome regions, focusing on their critical roles in viral RNA synthesis and gene expression.

RNA, Viral↗

PubMed2006

Distinct and overlapping sets of SUMO-1 and SUMO-2 target proteins revealed by quantitative proteomics.

The small ubiquitin-like modifier (SUMO) family in vertebrates includes three different family members that are conjugated as post-translational modifications to target proteins. SUMO-2 and -3 are nearly identical but differ substantially from SUMO-1. We used quantitative proteomics to investigate the target protein preferences of SUMO-1 and SUMO-2. HeLa cells were established that stably express His6-SUMO-1 or His6-SUMO-2. These cell lines and control HeLa cells were labeled with stable arginine isotopes, and His6-SUMOs were enriched from lysates using immobilized metal affinity chromatography. 53 SUMO-conjugated proteins were identified, including 44 novel SUMO targets. 25 proteins were preferentially conjugated to SUMO-1, 19 were preferentially conjugated to SUMO-2, and nine proteins were conjugated to both SUMO-1 and SUMO-2. SART1 was confirmed by immunoblotting to have both SUMO-1- and SUMO-2-linked forms at similar levels. SUMO-1 and SUMO-2 are thus shown to have distinct and overlapping sets of target proteins, indicating that SUMO-1 and SUMO-2 may have both redundant and non-redundant cellular functions. Interestingly, 14 of the 25 SUMO-1-conjugated proteins contain zinc fingers. Although both SUMO family members play roles in many cellular processes, our data show that sumoylation is strongly associated with transcription because nearly one-third of the identified target proteins are putative transcriptional regulators.

Chromatography, Affinity↗