Specific DNA Binding Research Articles

UV irradiation remodels the specificity landscape of transcription factors

Somatic mutations are highly enriched at transcription factor (TF) binding sites, with the strongest trend being observed for ultraviolet light (UV)-induced mutations in melanomas. One of the main mechanisms proposed for this hypermutation pattern is the inefficient repair of UV lesions within TF-binding sites, caused by competition between TFs bound to these lesions and the DNA repair proteins that must recognize the lesions to initiate repair. However, TF binding to UV-irradiated DNA is poorly characterized, and it is unclear whether TFs maintain specificity for their DNA sites after UV exposure. We developed UV-Bind, a high-throughput approach to investigate the impact of UV irradiation on protein-DNA binding specificity. Weapplied UV-Bind to ten TFs from eight structural families, and found that UV lesions significantly altered the DNA-binding preferences of all the TFs tested. The main effect was a decrease in binding specificity, but the precise effects and their magnitude differ across factors. Importantly, we found that despite the overall reduction in DNA-binding specificity in the presence of UV lesions, TFs can still compete with repair proteins for lesion recognition, in a manner consistent with their specificity for UV-irradiated DNA. In addition, for a subset of TFs, we identified a surprising but reproducible effect at certain nonconsensus DNA sequences, where UV irradiation leads to a high increase in the level of TF binding. These changes in DNA-binding specificity after UV irradiation, at both consensus and nonconsensus sites, have important implications for the regulatory and mutagenic roles of TFs in the cell.

Cooperative action of separate interaction domains promotes high-affinity DNA binding of Arabidopsis thaliana ARF transcription factors

The signaling molecule auxin coordinates many growth and development processes in plants, mainly through modulating gene expression. Transcriptional response is mediated by the family of auxin response factors (ARF). Monomers of this family recognize a DNA motif and can homodimerize through their DNA-binding domain (DBD), enabling cooperative binding to an inverted binding site. Most ARFs further contain a C-terminal PB1 domain that is capable of homotypic interactions and mediating interactions with Aux/IAA repressors. Given the dual role of the PB1 domain, and the ability of both DBD and PB1 domain to mediate dimerization, a key question is how these domains contribute to DNA-binding specificity and affinity. So far, ARF-ARF and ARF-DNA interactions have mostly been approached using qualitative methods that do not provide a quantitative and dynamic view on the binding equilibria. Here, we utilize a DNA binding assay based on single-molecule Förster resonance energy transfer (smFRET) to study the affinity and kinetics of the interaction of several Arabidopsis thaliana ARFs with an IR7 auxin-responsive element (AuxRE). We show that both DBD and PB1 domains of AtARF2 contribute toward DNA binding, and we identify ARF dimer stability as a key parameter in defining binding affinity and kinetics across AtARFs. Lastly, we derived an analytical solution for a four-state cyclic model that explains both the kinetics and the affinity of the interaction between AtARF2 and IR7. Our work demonstrates that the affinity of ARFs toward composite DNA response elements is defined by dimerization equilibrium, identifying this as a key element in ARF-mediated transcriptional activity.

Zinc binding of a Cys2His2-type zinc finger protein is enhanced by the interaction with DNA

Zinc finger proteins specifically recognize DNA sequences and, therefore, play a crucial role in living organisms. In this study the Zn(II)-, and DNA-binding of 1MEY#, an artificial zinc finger protein consisting of three finger units was characterized by multiple methods. Fluorimetric, circular dichroism and isothermal calorimetric titrations were applied to determine the accurate stability constant of a zinc finger protein. Assuming that all three zinc finger subunits behave identically, the obtained thermodynamic data for the Zn(II) binding were ΔHbinding site = − (23.5 − 28.0) kcal/mol (depending on the applied protonation state of the cysteines) and logβ’pH 7.4 = 12.2 ± 0.1, being similar to those of the CP1 consensus zinc finger peptide. The specific DNA binding of the protein can be characterized by logβ’pH 7.4 = 8.20 ± 0.08, which is comparable to the affinity of the natural zinc finger proteins (Sp1, WT1, TFIIIA) toward DNA. This value is ~ 1.9 logβ’ unit higher than those determined for semi- or nonspecific DNA binding. Competitive circular dichroism and electrophoretic mobility shift measurements revealed that the conditional stability constant characteristic for Zn(II) binding of 1MEY# protein increased by 3.4 orders of magnitude in the presence of its target DNA sequence.Graphical abstract

Dual specificity and target gene selection by the MADS-domain protein FRUITFULL.

How transcription factors attain their target gene specificity and how this specificity may be modulated, acquiring different regulatory functions through the development of plant tissues, is an open question. Here we characterized different regulatory roles of the MADS-domain transcription factor FRUITFULL (FUL) in flower development and mechanisms modulating its activity. We found that the dual role of FUL in regulating floral transition and pistil development is associated with its different in vivo patterns of DNA binding in both tissues. Characterization of FUL protein complexes by liquid chromatography-tandem mass spectrometry and SELEX-seq experiments shows that aspects of tissue-specific target site selection can be predicted by tissue-specific variation in the composition of FUL protein complexes with different DNA binding specificities, without considering the chromatin status of the target region. This suggests a role for dynamic changes in FUL TF complex composition in reshaping the regulatory functions of FUL during flower development.

DNA methylation entropy is associated with DNA sequence features and developmental epigenetic divergence.

Epigenetic information defines tissue identity and is largely inherited in development through DNA methylation. While studied mostly for mean differences, methylation also encodes stochastic change, defined as entropy in information theory. Analyzing allele-specific methylation in 49 human tissue sample datasets, we find that methylation entropy is associated with specific DNA binding motifs, regulatory DNA, and CpG density. Then applying information theory to 42 mouse embryo methylation datasets, we find that the contribution of methylation entropy to time- and tissue-specific patterns of development is comparable to the contribution of methylation mean, and methylation entropy is associated with sequence and chromatin features conserved with human. Moreover, methylation entropy is directly related to gene expression variability in development, suggesting a role for epigenetic entropy in developmental plasticity.

The F-box protein UFO controls flower development by redirecting the master transcription factor LEAFY to new cis-elements.

In angiosperms, flower development requires the combined action of the transcription factor LEAFY (LFY) and the ubiquitin ligase adaptor F-box protein, UNUSUAL FLORAL ORGANS (UFO), but the molecular mechanism underlying this synergy has remained unknown. Here we show in transient assays and stable transgenic plants that the connection to ubiquitination pathways suggested by the UFO F-box domain is mostly dispensable. On the basis of biochemical and genome-wide studies, we establish that UFO instead acts by forming an active transcriptional complex with LFY at newly discovered regulatory elements. Structural characterization of the LFY-UFO-DNA complex by cryo-electron microscopy further demonstrates that UFO performs this function by directly interacting with both LFY and DNA. Finally, we propose that this complex might have a deep evolutionary origin, largely predating flowering plants. This work reveals a unique mechanism of an F-box protein directly modulating the DNA binding specificity of a master transcription factor.

Non-specific vs. specific DNA binding free energetics of a transcription factor domain protein between search and recognition.

Transcription factor (TF) proteins are key in genetic regulation by locating specific sites on the genome. It has been proposed that an optimized search can be achieved by protein alternating between 3D diffusion in the bulk and 1D diffusion along DNA in the facilitated diffusion model. While undergoing 1D diffusion, the protein can switch from a “search” mode for fast diffusion along non-specific DNA, to a “recognition” mode for protein stable binding to specific DNA. Though it was regarded that protein conformation transitions enable the search to recognition transitions, it was recently noticed that for a small TF domain protein, re-orientation of the protein on the DNA, without protein conformational changes, happens between the non-specific and specific DNA binding. We further conducted all-atom molecular dynamics simulations on the TF domain protein bound to a nonspecific or specific DNA site, to energetically confirm that the protein-DNA binding free energetics between the two systems is consistent with the “search” and “recognition”. Using both the Jarzynski equality and umbrella sampling methods pulling protein away from DNA, we obtained protein binding free energetics and a free energy difference of about 10 kBT between the two systems. However, the binding free energy difference estimated from experimental measurements was about 4 kBT on 15-bp DNA. We suggest that the discrepancy between the experimental and computational measurements can be due to DNA sequences flanking the 6-bp central specific and nonspecific binding sites. Such a proposal was also investigated through a highly simplified spherical protein model along with stochastic simulations, which well supported that flanking DNA sequences impact on overall protein dissociation kinetics and therefore on measuring binding affinity variations with specific or non-specific DNA in the central binding site.

Insight into the intrinsically disordered transactivation domain of NFkB as a regulator of DNA binding using UV-induced unnatural amino acid crosslinking.

Within our vast genome, specific transcription factors like NFkB manage to find target sequences amongst overwhelming nonspecific options and help elicit timely cellular responses. A transcription factor's binding affinity for specific vs. nonspecific DNA must therefore be in a delicate balance to facilitate this search. Key in vitro experiments that described these binding affinities for the dominant NFkB dimer RelA/p50 were carried out with a truncated version of RelA - excluding a large intrinsically disordered transactivation domain (TAD) that was believed to be important for transcription activation through cofactor interactions but not impactful for DNA binding. This fit with a traditional view of protein domains acting as modular units. More recently, however, we discovered a novel function of the TAD: binding experiments revealed that the TAD's presence increased RelA/p50's affinity for DNA, with the strongest increase in binding affinity observed for non-specific DNA sequences lacking kB consensus sites. Here we follow up these findings with an investigation of the TAD's mechanism of action. We present preliminary results using an unnatural amino acid crosslinker (p-benzoyl-l-phenylalanine or pBpF) in combination with mass spectrometry to probe TAD interactions that lead to the observed changes in DNA binding affinity and specificity. pBpF is incorporated into specific positions in the TAD using AMBER codon suppression, and crosslinking is initiated by UV light. Interactions are analyzed by mass spectrometry to identify key sites of contact between the TAD and the natively folded Rel-Homology Domain.

Beyond the DNA binding domain: How do intrinsically disordered regions within transcription factors impact DNA binding specificity?

Transcription factors (TFs) are proteins that regulate transcription via site-specific binding to regulatory regions of the genome. Canonical models assume this site-specific binding is dictated by the DNA binding domain (DBD) alone and that the remainder of the protein does not impact specificity. This assumption underlies many assays commonly used in molecular biology (e.g. yeast-2-hybrid screens) and a longstanding focus on profiling TF-DNA binding specificity experimentally using truncated DBD-only constructs. However, recent surprising in vivo evidence suggested that specificity is heavily influenced by the presence or absence of intrinsically disordered protein regions outside of the DBD (non-DBDs). Further, this suggests that models of TF-DNA binding must incorporate mechanisms by which non-DBDs impact binding specificity in order to accurately predict this binding. Here, we systematically investigate in vitro the potential biophysical mechanisms by which non-DBDs could alter specificity. Using a suite of high-throughput in vitro microfluidics binding assays (BET-seq and MITOMI), we systematically quantify binding energies for wildtype, truncation, and DBD-swap constructs derived from S. cerevisiae TF paralogs, Msn2, Msn4, and Com2, the non-DBDs of which were shown to affect binding specificity in vivo. By measuring binding of each construct to a library of 213,496 sequences tiling across all S. cerevisiae promoters as well as sequences systematically varying the known consensus motif and flanking nucleotides, we quantify impacts of non-DBDs on TF-DNA binding specificity landscapes, elucidating the mechanisms by which TFs find and bind target genomic loci.

A multimorphic mutation in IRF4 causes human autosomal dominant combined immunodeficiency.

Interferon regulatory factor 4 (IRF4) is a transcription factor (TF) and key regulator of immune cell development and function. We report a recurrent heterozygous mutation in IRF4, p.T95R, causing an autosomal dominant combined immunodeficiency (CID) in seven patients from six unrelated families. The patients exhibited profound susceptibility to opportunistic infections, notably Pneumocystis jirovecii, and presented with agammaglobulinemia. Patients' B cells showed impaired maturation, decreased immunoglobulin isotype switching, and defective plasma cell differentiation, whereas their T cells contained reduced TH17 and TFH populations and exhibited decreased cytokine production. A knock-in mouse model of heterozygous T95R showed a severe defect in antibody production both at the steady state and after immunization with different types of antigens, consistent with the CID observed in these patients. The IRF4T95R variant maps to the TF's DNA binding domain, alters its canonical DNA binding specificities, and results in a simultaneous multimorphic combination of loss, gain, and new functions for IRF4. IRF4T95R behaved as a gain-of-function hypermorph by binding to DNA with higher affinity than IRF4WT. Despite this increased affinity for DNA, the transcriptional activity on IRF4 canonical genes was reduced, showcasing a hypomorphic activity of IRF4T95R. Simultaneously, IRF4T95R functions as a neomorph by binding to noncanonical DNA sites to alter the gene expression profile, including the transcription of genes exclusively induced by IRF4T95R but not by IRF4WT. This previously undescribed multimorphic IRF4 pathophysiology disrupts normal lymphocyte biology, causing human disease.

Biochemical characterization and mechanistic insight of the family IV uracil DNA glycosylase from Sulfolobus islandicus REY15A

Uracil DNA glycosylase (UDG) can remove uracil from DNA, thus playing an essential role in maintaining genomic stability. Family IV UDG members are mostly widespread in hyperthermophilic Archaea and bacteria. In this work, we characterized the family IV UDG from the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A (Sis-UDGIV) biochemically, and dissected the roles of nine conserved residues in uracil excision by mutational analyses. Biochemical data demonstrate that Sis-UDGIV displays maximum efficiency for uracil excision at 50 °C ~ 70 °C and at pH 7.0–9.0. Additionally, the enzyme has displays a weak activity without a divalent metal ion, but maximum activity with Mg2+. Our mutational analyses show that residues E48 and F55 in Sis-UDGIV are essential for uracil removal, and residues E48, F55, R87, R92 and K146 are responsible for binding DNA. Importantly, we systemically revealed the roles of four conserved cysteine residues C14, C17, C86 and C102 in Sis-UDGIV that are required for being ligands of FeS cluster in maintaining the overall protein conformation and stability by circular dichroism analyses. Overall, our work has provided insights into biochemical function and DNA-binding specificity of archaeal family IV UDGs.

Evolution of nanobodies specific for BCL11A

Transcription factors (TFs) control numerous genes that are directly relevant to many human disorders. However, developing specific reagents targeting TFs within intact cells is challenging due to the presence of highly disordered regions within these proteins. Intracellular antibodies offer opportunities to probe protein function and validate therapeutic targets. Here, we describe the optimization of nanobodies specific for BCL11A, a validated target for the treatment of hemoglobin disorders. We obtained first-generation nanobodies directed to a region of BCL11A comprising zinc fingers 4 to 6 (ZF456) from a synthetic yeast surface display library, and employed error-prone mutagenesis, structural determination, and molecular modeling to enhance binding affinity. Engineered nanobodies recognized ZF6 and mediated targeted protein degradation (TPD) of BCL11A protein in erythroid cells, leading to the anticipated reactivation of fetal hemoglobin (HbF) expression. Evolved nanobodies distinguished BCL11A from its close paralog BCL11B, which shares an identical DNA-binding specificity. Given the ease of manipulation of nanobodies and their exquisite specificity, nanobody-mediated TPD of TFs should be suitable for dissecting regulatory relationships of TFs and gene targets and validating therapeutic potential of proteins of interest.

Genetic difference between two Schistosoma japonicum isolates with contrasting cercarial shedding patterns revealed by whole genome sequencing

Schistosoma japonicum is one of the major infectious agents of human schistosomiasis, mainly endemic in China and the Philippines. We have previously reported the finding of two schistosome isolates, each with a different cercarial emergence pattern adapted to their different hosts. However, there are currently no whole-genome sequencing studies to investigate the underlining genetics of the adaptive traits. We sampled schistosomes in 2013 and 2020 from a hilly area Shitai (ST) and a marshland area Hexian (HX) of Anhui, China. Ten to 15 male or female adult worms from each site/year were sent for whole genome sequencing. Genetics were analyzed, and selection signals along genomes were detected. Gene enrichment analysis was performed for the genome regions under selection. The results revealed considerable genetic differentiation between the two isolates. The genome “windows” affected by natural selection were fewer in ST (64 windows containing 78 genes) than in HX (318 windows containing 276 genes). Twelve significantly enriched genes were identified in ST, but none in HX. These genes were mainly related to specific DNA binding and intercellular signaling transduction. Some functional region changes identified along the genome of the hilly schistosome may be related to its unique late afternoon cercarial emergence.

Structural characterization of protein-DNA complexes using small angle X-ray scattering (SAXS) with contrast variation.

Molecular and atomic level characterization of transcription factor (TF)-DNA complexes is critical for understanding DNA-binding specificity and potentially structural changes that may occur in protein and/or DNA upon complex formation. Often TFs are large, multidomain proteins or contain disordered regions that contribute to DNA recognition and/or binding affinity but are difficult to structurally characterize due to their high molecular weight and intrinsic flexibility. This results in challenges to obtaining high resolution structural information using Nuclear Magnetic Resonance (NMR) spectroscopy due to the relatively large size of the protein-DNA complexes of interest or macromolecular crystallography due to the difficulty in obtaining crystals of flexible proteins. Small angle X-ray scattering (SAXS) offers a complementary method to NMR and X-ray crystallography that allows for low-resolution structural characterization of protein, DNA, and protein-DNA complexes in solution over a greater size range and irrespective of interdomain flexibility and/or disordered regions. One important caveat to SAXS data interpretation, however, has been the inability to distinguish between scattering coming from the protein versus DNA component of the complex of interest. Here, we present a protocol using contrast variation via increasing sucrose concentrations to distinguish between protein and DNA using the model protein bovine serum albumin (BSA) and DNA and the LUX ARRYTHMO TF-DNA complex. Examination of the scattering curves of the components individually and in combination with contrast variation allows the differentiation of protein and DNA density in the derived models. This protocol is designed for use on high flux SAXS beamlines with temperature-controlled sample storage and sample exposure units.

Estimating DNA-Binding Specificities of Transcription Factors Using SELEX-Seq.

Here we provide an updated protocol forthe Systematic Evolution of Ligands followed by massively parallel sequencing (SELEX-seq) methodto study protein-DNA interaction specificities. This in vitro method is used to characterize DNA-binding specificities of transcription factors (TFs). The procedureis based on cycles of immunoprecipitation of protein-DNA complexes, starting with a randomized DNA library of defined fragment length, followed by massively parallel sequencing. The updated protocolincludes aspects ofexperimental design and procedure as well as basicinstructions on data analysis.

Coupling of binding and differential subdomain folding of the intrinsically disordered transcription factor CREB.

The cyclic AMP response element binding protein (CREB) contains a basic leucine zipper motif (bZIP) that forms a coiled coil structure upon dimerization and specific DNA binding. Although this state is well characterized, key features of CREB bZIP binding and folding are not well understood. We used single-molecule Förster resonance energy transfer (smFRET) to probe conformations of CREB bZIP subdomains. We found differential folding of the basic region and leucine zipper in response to different binding partners; a strong and previously unreported DNA-independent dimerization affinity; folding upon binding to nonspecific DNA; and evidence of long-range interdomain interactions in full-length CREB that modulate DNA binding. These studies provide new insights into DNA binding and dimerization and have implications for CREB function.

DDB2 represses epithelial-to-mesenchymal transition and sensitizes pancreatic ductal adenocarcinoma cells to chemotherapy.

Damage specific DNA binding protein 2 (DDB2) is an UV-indiced DNA damage recognition factor and regulator of cancer development and progression. DDB2 has dual roles in several cancers, either as an oncogene or as a tumor suppressor gene, depending on cancer localization. Here, we investigated the unresolved role of DDB2 in pancreatic ductal adenocarcinoma (PDAC). The expression level of DDB2 in pancreatic cancer tissues and its correlation with patient survival were evaluated using publicly available data. Two PDAC cell models with CRISPR-modified DDB2 expression were developed: DDB2 was repressed in DDB2-high T3M4 cells (T3M4 DDB2-low) while DDB2 was overexpressed in DDB2-low Capan-2 cells (Capan-2 DDB2-high). Immunofluorescence and qPCR assays were used to investigate epithelial-to-mesenchymal transition (EMT) in these models. Migration and invasion properties of the cells were also determined using wound healing and transwell assays. Sensitivity to 5-fluorouracil (5-FU), oxaliplatin, irinotecan and gemcitabine were finally investigated by crystal violet assays. DDB2 expression level was reduced in PDAC tissues compared to normal ones and DDB2-low levels were correlated to shorter disease-free survival in PDAC patients. DDB2 overexpression increased expression of E-cadherin epithelial marker, and decreased levels of N-cadherin mesenchymal marker. Conversely, we observed opposite effects in DDB2 repression and enhanced transcription of SNAIL, ZEB1, and TWIST EMT transcription factors (EMT-TFs). Study of migration and invasion revealed that these properties were negatively correlated with DDB2 expression in both cell models. DDB2 overexpression sensitized cells to 5-fluorouracil, oxaliplatin and gemcitabine. Our study highlights the potential tumor suppressive effects of DDB2 on PDAC progression. DDB2 could thus represent a promising therapeutic target or biomarker for defining prognosis and predicting chemotherapy response in patients with PDAC.

The miRNA125a-5p and miRNA125b-1-5p cluster induces cell invasion by down-regulating DDB2-reduced epithelial-to-mesenchymal transition (EMT) in colorectal cancer

MicroRNA (miRNA) is a kind of non-coding RNA that regulates gene expression and is involved in tumor development. MiRNA-125 is reportedly aberrantly expressed in colorectal cancer tissue; however, its potential function and underlying mechanism remain unclear. The present study aimed to investigate the expression level and potential role of the miRNA-125 family in the invasion and migration of colorectal cancer. To further understand the role of the miRNA-125 family in metastatic colorectal cancer, we overexpressed miRNA-125 in the SW480 cell line by transfection with the miRNA-125 family mimics or a sponge. Methyl thiazolyl tetrazolium (MTT) assay was performed to identify the effect of the miRNA-125 family on cell proliferation, and a Transwell filter assay was used to detect the role of the miRNA-125 family in migration and invasion. A luciferase assay was carried out to confirm the binding site of miRNA-125 and the target gene, damage specific DNA binding protein 2 (DDB2). Western blot was applied to detect the expression levels of DDB2 and the markers of epithelial-to-mesenchymal transition (EMT) in colorectal cancer cells. The real-time polymerase chain reaction (PCR) results showed that miR-125a-5p and miR-125b-1-5p were up-regulated in metastatic colorectal cancer tissues. The Transwell filter assay results appeared that miR-125a-5p and miR-125b-1-5p could promote the invasion and migration of colorectal cancer cells. The luciferase assay data confirmed the binding site of miR-125a-5p and miR-125b-1-5p on the 3' untranslated region (3'UTR) of DDB2 messenger RNA (mRNA). The real-time PCR and Western blot results indicated that miR-125a-5p and miR-125b-1-5p could regulate the expression levels of DDB2 and EMT markers, and lower DDB2 expression was observed in metastatic tissues. Our findings illustrated that miRNA125a-5p and miRNA125b-1-5p could reduce the expression of DDB2 by binding to the 3'UTR region, and then regulate the expression levels of EMT markers, leading to the enhanced invasion and metastasis of colorectal cancer cells. Thus, miRNA125a-5p and miRNA125b-1-5p might be novel markers of colorectal cancer migration and potential therapeutic targets to treat metastatic colorectal cancer patients.

Identification and Functional Analysis of bZIP Genes in Cotton Response to Drought Stress

The basic leucine zipper (bZIP) transcription factors, which harbor a conserved bZIP domain composed of two regions, a DNA-binding basic region and a Leu Zipper region, operate as important switches of transcription networks in eukaryotes. However, this gene family has not been systematically characterized in cotton (Gossypium hirsutum). Here, we identified 197 bZIP family members in cotton. The chromosome distribution pattern indicates that the GhbZIP genes have undergone 53 genome-wide segmental and 7 tandem duplication events which contribute to the expansion of the cotton bZIP family. Phylogenetic analysis showed that cotton GhbZIP proteins cluster into 13 subfamilies, and homologous protein pairs showed similar characteristics. Inspection of the DNA-binding basic region and leucine repeat heptads within the bZIP domains indicated different DNA-binding site specificities as well as dimerization properties among different groups. Comprehensive expression analysis indicated the most highly and differentially expressed genes in root and leaf that might play significant roles in cotton response to drought stress. GhABF3D was identified as a highly and differentially expressed bZIP family gene in cotton leaf and root under drought stress treatments that likely controls drought stress responses in cotton. These data provide useful information for further functional analysis of the GhbZIP gene family and its potential application in crop improvement.

Dissection of Functional Domains of Orc1-2, the Archaeal Global DNA Damage-Responsive Regulator

Orc1-2 is a non-initiator ortholog of archaeal/eukaryotic Orc1 proteins, which functions as a global regulator in DNA damage-responsive (DDR) expression. As for Orc1 initiators, the DDR regulator harbors an AAA+ ATPase domain, an Initiator-Specific Motif (ISM) and a winged-helix (wH) DNA-binding domain, which are also organized in a similar fashion. To investigate how Orc1-2 mediates the DDR regulation, the orc1-2 mutants inactivating each of these functional domains were constructed with Saccharolobus islandicus and genetically characterized. We found that disruption of each functional domain completely abolished the DDR regulation in these orc1-2 mutants. Strikingly, inactivation of ATP hydrolysis of Orc1-2 rendered an inviable mutant. However, the cell lethality can be suppressed by the deficiency of the DNA binding in the same protein, and it occurs independent of any DNA damage signal. Mutant Orc1-2 proteins were then obtained and investigated for DNA-binding in vitro. This revealed that both the AAA+ ATPase and the wH domains are involved in DNA-binding, where ISM and R381R383 in wH are responsible for specific DNA binding. We further show that Orc1-2 regulation occurs in two distinct steps: (a) eliciting cell division inhibition at a low Orc1-2 content, and this regulation is switched on by ATP binding and turned off by ATP hydrolysis; any failure in turning off the regulation leads to growth inhibition and cell death; (b) activation of the expression of DDR gene encoding DNA repair proteins at an elevated level of Orc1-2.