DNA Termini Research Articles

Mre11-Rad50: the DNA end game.

The Mre11-Rad50-(Nbs1/Xrs2) complex is an evolutionarily conserved factor for the repair of DNA double-strand breaks and other DNA termini in all kingdoms of life. It is an intricate DNA associated molecular machine that cuts, among other functions, a large variety of free and obstructed DNA termini for DNA repair by end joining or homologous recombination, yet leaves undamaged DNA intact. Recent years have brought progress in both the structural and functional analyses of Mre11-Rad50 orthologs, revealing mechanisms of DNA end recognition, endo/exonuclease activities, nuclease regulation and DNA scaffolding. Here, I review our current understanding and recent progress on the functional architecture Mre11-Rad50 and how this chromosome associated coiled-coil ABC ATPase acts as DNA topology specific endo-/exonuclease.

Cloning and Overexpression of Staphylococcal Enterotoxin B gene synthetic (SEBsyn) in pET-28a: Pre-development bacterial-toxin therapy for cancer

Staphylococcal enterotoxin B (SEB) from Staphylococcus aureus is a potential therapeutic agent for cancer. SEB can activate the immune response, which could induce apoptosis of various cancer cells. This study aimed to design and produce a recombinant SEB protein using Escherichia coli BL21(DE3) expression system. We designed and optimized the codon of the inserted gene for E. coli and then transformed it into E. coli BL21(DE3). The transformants were verified by selective media containing kanamycin followed by colony PCR with T7 promoter and T7 terminator primers and DNA sequencing. The results showed that the SEBsyn encoding gene could be synthesized and cloned into pET-28a(+) expression plasmid. This recombinant plasmid that carried the SEB encoding gene (pET-28a_SEBsyn) was successfully transformed into E. coli BL21(DE3). The amplicons of colony PCR visualized in 2% agarose gel showed that transformants carrying the recombinant plasmid had an inserted gene length of about 1 kb. Gene inserts sequence verification by DNA sequencing resulted in 1,148 bp sequence consensus. The blastx analysis showed that it had the best hit with enterotoxin B in the NCBI database accession id CAC6284025.1. The recombinant SEB protein was also successfully overexpressed in E.coli BL21(DE3) with 0.1 mM IPTG. Our recombinant SEB protein was dominantly insoluble in the inclusion body; however, some SEB protein was expressed as soluble. The molecular weight of the target protein is about 29 kDa.

Fluorescently labeled human apurinic/apyrimidinic endonuclease APE1 reveals effects of DNA polymerase β on the APE1–DNA interaction

The base excision repair (BER) pathway involves sequential action of DNA glycosylases and apurinic/apyrimidinic (AP) endonucleases to incise damaged DNA and prepare DNA termini for incorporation of a correct nucleotide by DNA polymerases. It has been suggested that the enzymatic steps in BER include recognition of a product–enzyme complex by the next enzyme in the pathway, resulting in the “passing-the-baton” model of transfer of DNA intermediates between enzymes. To verify this model, in this work, we aimed to create a suitable experimental system. We prepared APE1 site-specifically labeled with a fluorescent reporter that is sensitive to stages of APE1–DNA binding, of formation of the catalytic complex, and of subsequent dissociation of the enzyme–product complex. Interactions of the labeled APE1 with various model DNA substrates (containing an abasic site) of varied lengths revealed that the enzyme remains mostly in complex with the DNA product. By means of the fluorescently labeled APE1 in combination with a stopped-flow fluorescence assay, it was found that Polβ stimulates both i) APE1 binding to an abasic-site–containing DNA duplex with the formation of a catalytically competent complex and ii) the dissociation of APE1 from its product. These findings confirm DNA-mediated coordination of APE1 and Polβ activities and suggest that Polβ is the key trigger of the DNA transfer between the enzymes participating in initial steps of BER.

Mammalian N1-adenosine PARylation is a reversible DNA modification

Poly-ADP-ribosylation (PARylation) is regarded as a protein-specific modification. However, some PARPs were recently shown to modify DNA termini in vitro. Here, we use ultrasensitive mass spectrometry (LC-MS/MS), anti-PAR antibodies, and anti-PAR reagents to show that mammalian DNA is physiologically PARylated and to different levels in primary tissues. Inhibition of PAR glycohydrolase (PARG) increases DNA PARylation, supporting that the modification is reversible. DNA PARylation requires PARP1 and in vitro PARP1 PARylates single-stranded DNA, while PARG reverts the modification. DNA PARylation occurs at the N1-position of adenosine residues to form N1-Poly(ADP-ribosyl)-deoxyadenosine. Through partial hydrolysis of mammalian gDNA we identify PAR-DNA via the diagnostic deamination product N1-ribosyl-deoxyinosine to occur in vivo. The discovery of N1-adenosine PARylation as a DNA modification establishes the conceptual and methodological framework to elucidate its biological relevance and extends the role of PARP enzymes.

Molecular basis for processing of topoisomerase 1-triggered DNA damage by Apn2/APE2.

Topoisomerase 1 (Top1) incises DNA containing ribonucleotides to generate complex DNA lesions that are resolved by APE2 (Apn2 in yeast). How Apn2 engages and processes this DNA damage is unclear. Here, we report X-ray crystal structures and biochemical analysis of Apn2-DNA complexes to demonstrate how Apn2 frays and cleaves 3' DNA termini via a wedging mechanism that facilitates 1-6 nucleotide endonucleolytic cleavages. APN2 deletion and DNA-wedge mutant Saccharomyces cerevisiae strains display mutator phenotypes, cell growth defects, and sensitivity to genotoxic stress in a ribonucleotide excision repair (RER)-defective background harboring a high density of Top1-incised ribonucleotides. Our data implicate a wedge-and-cut mechanism underpinning the broad-specificity Apn2 nuclease activity that mitigates mutagenic and genome instability phenotypes caused by Top1 incision at genomic ribonucleotides incorporated by DNA polymerase epsilon.

Improving bacterial genome assembly using a test of strand orientation.

The complexity of genome assembly is due in large part to the presence of repeats. In particular, large reverse-complemented repeats can lead to incorrect inversions of large segments of the genome. To detect and correct such inversions in finished bacterial genomes, we propose a statistical test based on tetranucleotide frequency (TNF), which determines whether two segments from the same genome are of the same or opposite orientation. In most cases, the test neatly partitions the genome into two segments of roughly equal length with seemingly opposite orientations. This corresponds to the segments between the DNA replication origin and terminus, which were previously known to have distinct nucleotide compositions. We show that, in several cases where this balanced partition is not observed, the test identifies a potential inverted misassembly, which is validated by the presence of a reverse-complemented repeat at the boundaries of the inversion. After inverting the sequence between the repeat, the balance of the misassembled genome is restored. Our method identifies 31 potential misassemblies in the NCBI database, several of which are further supported by a reassembly of the read data. A github repository is available at https://github.com/gcgreenberg/Oriented-TNF.git. Supplementary data are available at Bioinformatics online.

Abstract LB110: Twist pan-cancer synthetic reference materials for cell-free DNA (cfDNA) assay development

Abstract Liquid biopsies hold great promise in reducing the burden of the disease of cancer on patients. Early detection of cancer via the cell free DNA represents the broadest vision and requires highly specificity. Ongoing disease monitoring is another important application of liquid biopsy and requires high sensitivity. Assay development requires analytical validation, and assays must be monitored for ongoing performance. These assays demand reference materials at specific variant allele frequencies for the assessment of specificity and sensitivity. Previously, obtaining reference materials was a laborious process of finding or engineering mutant cell lines. Here, we describe the design, manufacture, and quality control of a synthetic reference standard: the Twist Pan-Cancer Reference Standards, developed using Twist’s proprietary DNA printing platform to include a wide selection of both common and rare cancer targets for analytical validation. The reference material closely mimics the size distribution and content of cell-free DNA, with a primary peak at 167 bp and a secondary peak at 334 bp. The background cfDNA is derived from a single donor and highly characterized through NGS. The reference standard includes over 400 variant sites across 84 genes, including literature-curated, clinically-relevant variant sites. Additional panel-wide variants were included to aid in troubleshooting capture panels. The variant DNA is synthetically printed at 167 bp ± 5 bp and tiles over the site with extensive overlap, providing diversity of DNA termini relative to the position of the site of variation. Variant sites were printed and quantified independently so they can be pooled uniformly. NGS quality controls are implemented at multiple steps to ensure quality. The final products are available in a variant allele frequency (VAF) dilution series. The dilution series is quality controlled using droplet digital PCR (ddPCR) to ensure precision of variant allele frequencies. The Twist Pan Cancer Reference Standards provide a valuable solution for NGS-based assay developers seeking reference materials for a wide array of cancer variants, advancing adoption of liquid biopsy tests toward routine clinical practice. Citation Format: Patrick D. Cherry. Twist pan-cancer synthetic reference materials for cell-free DNA (cfDNA) assay development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB110.

Abstract 2596: Impact of novel Ku-DNA binding inhibitors on the DNA DSBs-induced DNA damage response

Abstract The DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) double-strand break (DSB) repair pathway and the DNA damage response (DDR). Consequently, blocking DNA-PK kinase activity is a novel anti-cancer therapeutic strategy in combination with ionizing radiation (IR). Towards developing a new class of DNA-PK inhibitors, our laboratory has exploited the mechanism of DNA-PK activation which requires binding to DNA termini via the Ku 70/80 heterodimer. We have previously reported the development of Ku-DNA binding inhibitors (Ku-DBi’s) that act via this novel mechanism of action to inhibit DNA-PK catalytic kinase activity. Ku-DBi’s display nanomolar activity in vitro, possess cellular DNA-PK and NHEJ inhibitory activity and sensitize non-small cell lung cancer (NSCLC) cells to DSB generating chemotherapeutics bleomycin and etoposide. In this study, we demonstrate that pre-incubation of our novel Ku-DBi’s can potentiate the cellular effects of bleomycin and IR and also induce p53 phosphorylation through the activation of the ATM pathway, which is concomitant with a decrease in DNA-PKcs autophosphorylation events at the S2056 (pS2056) cluster. Using a combination of Western blot and immunofluorescence assays in the NSCLC NCI-H460 and A549 cell lines, Ku-DBi’s treatment in combination with DNA DSBs-inducing agents such as bleomycin and IR, showed a significant reduction of autophosphorylation events of DNA-PKcs at the S2056 cluster compared to DNA DSBs-inducing agent alone. In addition, analysis of phospho-ATM and phospho-p53 protein levels in these NSCLC cell lines, suggested activation of the ATM pathway as a function of Ku-DBi’s treatment followed by bleomycin, evidenced by an increase of phosphorylation of ATM at Ser1981, and a modest increase of p53 phosphorylation at Ser15. Our findings demonstrate that Ku-DBi’s block DNA-DSB dependent DNA-PKcs autophosphorylation, resulting in potentiating cellular sensitivity to bleomycin and IR, and a likely effect on the ATM-dependent signaling pathway. These data are consistent with Ku-DBi’s possessing a novel mechanism of action that abrogates autophosphorylation of DNA-PKcs to impact DSB repair and potentially DDR signaling, becoming in a promising approach as part of an anticancer therapeutic strategy in combination with DNA DSBs-inducing agents. Citation Format: Pamela L. Mendoza-Munoz, Navnath S. Gavande, Pamela S. VanderVere-Carozza, Katherine S. Pawelczak, Joseph R. Dynlacht, Joy E. Garrett, John J. Turchi. Impact of novel Ku-DNA binding inhibitors on the DNA DSBs-induced DNA damage response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2596.

TT-pocket/HIRAN: binding to 3'-terminus of DNA for recognition and processing of stalled replication forks.

Stalled replication forks need to be swiftly detected and protected from collapse and the cause for fork stall be removed to restore the active replication fork. In bacteria, stalled forks are recognized and stabilized by PriA, a DEXH-type helicase, which also facilitates reassembly of an active replication fork. A TT-pocket (three-prime terminus binding pocket) present in the N-terminal segment of PriA plays a crucial role in stabilization of the stalled forks by specifically binding to the 3$^\prime$-terminus of the nascent leading strand. Eukaryotic proteins, Rad5/HLTF, contain a TT-pocket related domain, HIRAN, that specifically binds to 3'-terminus of DNA and play a role in stalled fork processing. While the TT-pocket of PriA facilitates the formation of an apparently stable and immobile complex on a fork with a 3'-terminus at the fork junction, HIRAN of Rad5/HLTF facilitates fork regression by itself. A recent report shows that HIRAN can displace 3 nucleotides at the end of the duplex DNA, providing mechanistic insight into how stalled forks are reversed in eukaryotes. In this article, I will compare the roles of 3'-terminus binding domains in stalled fork processing in prokaryotes and in eukaryotes.

Interplay between symmetric arginine dimethylation and ubiquitylation regulates TDP1 proteostasis for the repair of topoisomerase I-DNA adducts.

Tyrosyl-DNA phosphodiesterase (TDP1) hydrolyzes the phosphodiester bond between a DNA 3' end and a tyrosyl moiety and is implicated in the repair of trapped topoisomerase I (Top1)-DNA covalent complexes (Top1cc). Protein arginine methyltransferase 5 (PRMT5) catalyzes arginine methylation of TDP1 at the residues R361 and R586. Here, we establish mechanistic crosstalk between TDP1 arginine methylation and ubiquitylation, which is critical for TDP1 homeostasis and cellular responses to Top1 poisons. We show that R586 methylation promotes TDP1 ubiquitylation, which facilitates ubiquitin/proteasome-dependent TDP1 turnover by impeding the binding of UCHL3 (deubiquitylase enzyme) with TDP1. TDP1-R586 also promotes TDP1-XRCC1 binding and XRCC1 foci formation at Top1cc-damage sites. Intriguingly, R361 methylation enhances the 3'-phosphodiesterase activity of TDP1 in real-time fluorescence-based cleavage assays, and this was rationalized using structural modeling. Together, our findings establish arginine methylation as a co-regulator of TDP1 proteostasis and activity, which modulates the repair of trapped Top1cc.

WS21.03 Low-cost chain termination DNA sequencing PCR reaction to diagnose CFTR gene mutations

WS21.03 Low-cost chain termination DNA sequencing PCR reaction to diagnose CFTR gene mutations

The N-terminal domain of the Agrobacterium tumefaciens telomere resolvase, TelA, regulates its DNA cleavage and rejoining activities

Linear replicons can be found in a minority of prokaryotic organisms, including Borrelia species and Agrobacterium tumefaciens. The problem with replicating the lagging strand end of linear DNAs is circumvented in these organisms by the presence of covalently closed DNA hairpin telomeres at the DNA termini. Telomere resolvases are enzymes responsible for generating these hairpin telomeres from a dimeric replication intermediate through a two-step DNA cleavage and rejoining reaction referred to as telomere resolution. It was previously shown that the agrobacterial telomere resolvase, TelA, possesses ssDNA annealing activity in addition to telomere resolution activity. The annealing activity derives, chiefly, from the N-terminal domain. This domain is dispensable for telomere resolution. In this study, we used activity analyses of an N-terminal domain deletion mutant, domain add back experiments, and protein–protein interaction studies and we report that the N-terminal domain of TelA is involved in inhibitory interactions with the remainder of TelA that are relieved by the binding of divalent metal ions. We also found that the regulation of telomere resolution by the N-terminal domain of TelA extends to suppression of inappropriate enzymatic activity, including hairpin telomere fusion (reaction reversal) and recombination between replicated telomeres to form a Holliday junction.

Human TDP1, APE1and TREX1 repair 3'-DNA-peptide/protein cross-links arising from abasic sites in vitro.

Histones and many other proteins react with abundant endogenous DNA lesions, apurinic/apyrimidinic (abasic, AP) sites and/or 3′-phospho-α,β-unsaturated aldehyde (3′-PUA), to form unstable but long-lived Schiff base DNA–protein cross-links at 3′-DNA termini (3′-PUA–protein DPCs). Poly (ADP-ribose) polymerase 1 (PARP1) cross-links to the AP site in a similar manner but the Schiff base is reduced by PARP1’s intrinsic redox capacity, yielding a stable 3′-PUA–PARP1 DPC. Eradicating these DPCs is critical for maintaining the genome integrity because 3′-hydroxyl is required for DNA synthesis and ligation. But how they are repaired is not well understood. Herein, we chemically synthesized 3′-PUA-aminooxylysine-peptide adducts that closely resemble the proteolytic 3′-PUA–protein DPCs, and found that they can be repaired by human tyrosyl-DNA phosphodiesterase 1 (TDP1), AP endonuclease 1 (APE1) and three-prime repair exonuclease 1 (TREX1). We characterized these novel repair pathways by measuring the kinetic constants and determining the effect of cross-linked peptide length, flanking DNA structure, and the opposite nucleobase. We further found that these nucleases can directly repair 3′-PUA–histone DPCs, but not 3′-PUA–PARP1 DPCs unless proteolysis occurs initially. Collectively, we demonstrated that in vitro 3′-PUA–protein DPCs can be repaired by TDP1, APE1, and TREX1 following proteolysis, but the proteolysis is not absolutely required for smaller DPCs.

Structural basis of chromatin regulation by histone variant H2A.Z.

The importance of histone variant H2A.Z in transcription regulation has been well established, yet its mechanism-of-action remains enigmatic. Conflicting evidence exists in support of both an activating and a repressive role of H2A.Z in transcription. Here we report cryo-electron microscopy (cryo-EM) structures of nucleosomes and chromatin fibers containing H2A.Z and those containing canonical H2A. The structures show that H2A.Z incorporation results in substantial structural changes in both nucleosome and chromatin fiber. While H2A.Z increases the mobility of DNA terminus in nucleosomes, it simultaneously enables nucleosome arrays to form a more regular and condensed chromatin fiber. We also demonstrated that H2A.Z’s ability to enhance nucleosomal DNA mobility is largely attributed to its characteristic shorter C-terminus. Our study provides the structural basis for H2A.Z-mediated chromatin regulation, showing that the increase flexibility of the DNA termini in H2A.Z nucleosomes is central to its dual-functions in chromatin regulation and in transcription.

Labeling the 5' Termini of DNA with Bacteriophage T4 Polynucleotide Kinase.

The removal of 5' phosphates from nucleic acids with phosphatases and their readdition in radiolabeled form by bacteriophage T4 polynucleotide kinase is a widely used technique for generating 32P-labeled probes. When the reaction is performed efficiently, 40%-50% of the protruding 5' termini in the reaction becomes radiolabeled. However, the specific activity of the resulting probes is not as high as that obtained by other radiolabeling methods because only one radioactive atom is introduced per DNA molecule. Nevertheless, the availability of [γ-32P]ATP with specific activities in the 3000-7000 Ci/mmol range allows the synthesis of probes suitable for many purposes. This protocol includes procedures for labeling the 5' ends of dsDNA and oligonucleotides.

Surface plasmon resonance biosensor for the detection of miRNAs by combining the advantages of homogeneous reaction and heterogeneous detection

The hybridization and enzymolysis reactions for nucleic acid detection were carried out on the chip surface in the traditional surface plasmon resonance (SPR) biosensors. Herein, we proposed an innovative method for microRNA (miRNA) detection in which the hybridization-enzymolysis recycling reactions were performed in solution. Duplex-specific nuclease (DSN) and streptavidin-modified gold nanoparticles (SA-AuNPs) were employed for enhancing the assay sensitivity. In the absence of miRNA, the biotinylated DNA probe (bio-DNA-bio, biotin tags at both the 3′ and 5′ termini of DNA) was attached to the SA-modified chip through the SA-biotin binding, allowing the capture of SA-AuNPs with the same interaction. As a result, a larger SPR signal was attained. However, in the presence of miRNA, bio-DNA-bio hybridized with miRNA was digested by DSN. In this process, the miRNA strand remained intact and participated in the next hybridization-enzymolysis recycling process. Thus, one miRNA could promote the hydrolysis of many bio-DNA-bio probes and allow the generation of numerous bio-DNA fragments. Meanwhile, the produced bio-DNA competed with the undigested bio-DNA-bio to bind SA on the chip surface. The digestion of bio-DNA-bio and the competitive binding between bio-DNA-bio and bio-DNA led to the attachment of fewer SA-AuNPs and then smaller SPR signals. The change in SPR signal at the concentration as low as 1 fM miRNA has been readily determined. The strategy possessed the advantageous properties of simple operation, fast response, high sensitivity and excellent specificity, serving as a viable means for the fabrication of novel sensing platforms.

Sensitive and Label-free Detection of Bacteria in Osteomyelitis through Exo III-Assisted Cascade Signal Amplification.

Rapid and sensitive pathogenic bacterial identification and isolation from complicated clinical specimens are of great importance for the early diagnosis and prevention of osteomyelitis. Herein, we proposed a novel methicillin-resistant Staphylococcus aureus (MRSA) detection strategy through two specially designed streptavidin magnetic bead-based probes, including a capture probe and a report probe. In detail, the capture probe takes the responsibility to specially bind with the surface protein of MRSA and leads to the liberation of the promoter which could subsequently initiate report probe-based signal amplification. Afterward, the hybridization of the promoter probe with the report probe could then transform the protruding 3′ terminus of template DNA in the report probe into a blunt end. With the assistance of Exo III, the template could be digested to liberate the promoter to form a recycle and to liberate the biprobe to induce the following rolling circle amplification (RCA)-based signal amplification. Through the integration of the Exo III-assisted recycle and RCA-based signal amplification, the proposed method exhibited a favorable detection performance.

Abstract PO-023: Impact of a novel Ku-DNA binding inhibitor on the IR-induced DNA damage response

Abstract The DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) double stand break (DSB) repair pathway and the DNA damage response (DDR). Consequently, blocking DNA-PK kinase activity is a novel anti-cancer therapeutic strategy in combination with ionizing radiation (IR). Towards developing a series of a new class of DNA-PK inhibitors, our laboratory has exploited the mechanism of DNA-PK activation which requires binding to DNA termini via the Ku 70/80 heterodimer. We recently reported the development of Ku-DNA binding inhibitors (Ku-DBi’s) that act via this novel mechanism of action to inhibit DNA-PK catalytic kinase activity. Ku-DBi’s display nanomolar activity in vitro, possess cellular DNA-PK and NHEJ inhibitory activity and sensitize non-small cell lung cancer (NSCLC) cells to chemotherapeutics bleomycin and etoposide. In this study, we demonstrate that pre-incubation of our novel Ku-DBi’s can potentiate the cellular effects of IR. We demonstrate that the increase in IR sensitivity is preceded by a decrease in DNA-PKcs autophosphorylation events at the S2056 (pS2056) cluster. Using a combination of Western blot and immunofluorescence assays in the NSCLC NCI-H460 cell line, Ku-DBi 245 treatment in combination with IR showed a significant reduction of phosphorylation of DNA-PKcs at S2056 compared to IR alone. Analysis of gH2A.X also showed an inhibition of the DDR as a function of 245 treatment, evidenced by reduction of S139 phosphorylation. These results demonstrate that Ku-DBi 245 blocks DNA-break dependent DNA-PKcs autophosphorylation, resulting in an increased in radiation sensitivity. These data are consistent with Ku-DBi’s possessing a novel mechanism of action that abrogates autophosphorylation of DNA-PKcs and DDR signaling as part of an anticancer therapeutic strategy in combination with IR. Citation Format: Pamela Mendoza-Munoz, Navnath S. Gavande, Pamela S. VanderVere-Carozza, Katherine S. Pawelczak, Joseph R. Dynlacht, Joy Garrett, John J. Turchi. Impact of a novel Ku-DNA binding inhibitor on the IR-induced DNA damage response [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-023.

Z-Ring-Associated Proteins Regulate Clustering of the Replication Terminus-Binding Protein ZapT in Caulobacter crescentus.

Regulated organization of the chromosome is essential for faithful propagation of genetic information. In the model bacterium Caulobacter crescentus, the replication terminus of the chromosome is spatially arranged in close proximity to the cytokinetic Z-ring during the cell cycle. Although the Z-ring-associated proteins ZapA and ZauP interact with the terminus recognition protein ZapT, the molecular functions of the complex that physically links the terminus and the Z-ring remain obscure. In this study, we found that the physical linkage helps to organize the terminus DNA into a clustered structure. Neither ZapA nor ZauP was required for ZapT binding to the terminus DNA, but clustering of the ZapT-DNA complexes over the Z-ring was severely compromised in cells lacking ZapA or ZauP. Biochemical characterization revealed that ZapT, ZauP, and ZapA interacted directly to form a highly ordered ternary complex. Moreover, multiple ZapT molecules were sequestered by each ZauP oligomer. Investigation of the functional structure of ZapT revealed that the C terminus of ZapT specifically interacted with ZauP and was essential for timely positioning of the Z-ring in vivo Based on these findings, we propose that ZauP-dependent oligomerization of ZapT-DNA complexes plays a distinct role in organizing the replication terminus and the Z-ring. The C termini of ZapT homologs share similar chemical properties, implying a common mechanism for the physical linkage between the terminus and the Z-ring in bacteria.IMPORTANCE Rapidly growing bacteria experience dynamic changes in chromosome architecture during chromosome replication and segregation, reflecting the importance of mechanisms that organize the chromosome globally and locally within a cell to maintain faithful transmission of genetic material across generations. During cell division in the model bacterium Caulobacter crescentus, the replication terminus of the chromosome is physically linked to the cytokinetic Z-ring at midcell. However, the functions of this physical linkage are not fully understood. We adopted biochemical and cell-biological techniques to characterize the linkage, including the terminus-binding protein ZapT and the Z-ring-associated protein ZauP. We obtained evidence that the Z-ring organizes the chromosome terminus into a compact structure at midcell via specific interaction between ZapT and ZauP oligomers. Because these proteins are conserved in diverse Gram-negative bacteria, our findings highlight a novel and conserved role for the linker complex in regulated organization of the chromosome terminus.

Elemental composition control of gold-titania nanocomposites by site-specific mineralization using artificial peptides and DNA

Biomineralization, the precipitation of various inorganic compounds in biological systems, can be regulated in terms of the size, morphology, and crystal structure of these compounds by biomolecules such as proteins and peptides. However, it is difficult to construct complex inorganic nanostructures because they precipitate randomly in solution. Here, we report that the elemental composition of inorganic nanocomposites can be controlled by site-specific mineralization by changing the number of two inorganic-precipitating peptides bound to DNA. With a focus on gold and titania, we constructed a gold-titania photocatalyst that responds to visible light excitation. Both microscale and macroscale observations revealed that the elemental composition of this gold-titania nanocomposite can be controlled in several ten nm by changing the DNA length and the number of peptide binding sites on the DNA. Furthermore, photocatalytic activity and cell death induction effect under visible light (>450 nm) irradiation of the manufactured gold-titania nanocomposite was higher than that of commercial gold-titania and titania. Thus, we have succeeded in forming titania precipitates on a DNA terminus and gold precipitates site-specifically on double-stranded DNA as intended. Such nanometer-scale control of biomineralization represent a powerful and efficient tool for use in nanotechnology, electronics, ecology, medical science, and biotechnology.