Klenow Fragment Of Polymerase Research Articles

Label-Free, Sensitive, and Versatile Colorimetric Method for Molecule Detection via the G-Quadruplex-Based Signal Quenching Strategy.

Signal amplification strategies have emerged as a prominent tool in the field of improving the detection sensitivity of small extracellular vesicles (sEVs). It is important to highlight that the utilization of signal quenching strategies is not commonly implemented. A detection technique for sEVs was established based on the unwinding of G-quadruplex using Klenow fragment polymerase (KF), which served as an inspiration for this study. This system is characterized by its simplicity and lack of labeling, making it an efficient approach for signal quenching. In the presence of sEVs, the CD63 aptamer in the capture@sMBs complex binds with the CD63 protein on the surface of sEVs to release trigger sequences, which were employed as a primer to mediate the DNA polymerase/endonuclease-assisted signal recycling. The signal recycling process produces numerous single-stranded DNA sequences that can bind to the toehold section of the G-quadruplex. This leads to the rupture of the G-quadruplex structure and the subsequent deactivation of a DNAzyme generated by the G-quadruplex structure and hemin, thereby inhibiting its biological catalytic function. Consequently, the G-quadruplex structure would undergo a transformation to a duplex structure, leading to the emergence of a discernible differential signal that can be noticed in a majority of instances, even without the aid of magnification devices. The decrease in the prominent signal allows for the efficient analysis of target sEVs, which exhibit a notably low detection limit. In addition to the detection of sEVs, the approach has also been utilized for the investigation of miRNA-21. The approach demonstrates a high level of selectivity and robustness in its capacity to differentiate between target miRNA and base-mismatched miRNA as well as other miRNA families. This statement suggests that the assay holds significant promise for use in biochemical research and clinical diagnosis.

A novel biosensor based on antibody controlled isothermal strand displacement amplification (ACISDA) system

The overuse of antibiotics has aroused widespread concern in recent decades. Their residues in food and environment may pose potential risks to human health. Therefore, highly sensitive and rapid detection methods of antibiotics are urgently needed. Inspired by allosteric transcription factors (aTFs), we proposed a novel strategy for small molecules detection based on antibody controlled isothermal chain displacement amplification (ACISDA). A combination of nicking endonuclease, Klenow Fragment polymerase, specific antibody and a pair of antigen-labeled DNA regulate the synthesis of a G-quadruplex by isothermal chain displacement amplification. The presence of a target induces the antibody dissociation from the antigen-labeled DNA, which induces the synthesis of a G-quadruplex, and a fluorescent signal is produced by thioflavine T (ThT) binding to G-quadruplex. To test this notion, norfloxacin-conjugated DNA (named Primer-NOR) was prepared and ACISDA system was established combining with anti-norfloxacin antibody. This system could detect norfloxacin in a linear range of 0.1 ∼ 500 ng/mL with detection limit of 0.04 ng/mL, and this system could be applied to the detection of norfloxacin in real samples with good performance. Meanwhile, this system could also realize washing-free, immobilization-free and “ready-to-use”, and could be used for other small molecules quickly by replacing the antigen-labeled DNA and specific antibody.

A highly sensitive fluorescence method for the detection of T4 polynucleotide kinase phosphatase based on polydopamine nanotubes

T4 polynucleotide kinase phosphatase (T4 PNKP) plays a critical role in various cellular events, such as DNA damage repair, replication, and recombination. Here, we have described a novel biosensor to detect the activity of T4 PNKP based on polydopamine nanotubes (PDANTs) mediated fluorescence resonance energy transfer (FRET). A FAM-labelled (6-carboxyl-fluorescein) hairpin DNA probe with 3′-phosphoryl terminal was designed as the substrate for T4 PNKP. With the addition of PDANTs, the fluorescence of FAM-labelled hairpin DNA probe could be quenched because of the high adsorption of hairpin DNA on PDANTs. When T4 PNKP dephosphorylated the DNA probe, a double-stranded DNA (dsDNA) product was obtained by Klenow fragment polymerase (KF polymerase) on its 3′-hydroxyl terminal, which could retain most of the fluorescence due to the week adsorption of dsDNA on PDANTs. The developed method demonstrates the sensitivity for T4 PNKP assay in the range from 0.05 to 1.5 U mL−1 with the detection limit of 0.005 U mL−1, which endows the proposed strategy with high enough sensitivity for practical detection in cell lysates. With the advantages mentioned above, this novel sensitive strategy has the potential in the study of DNA damage repair mechanisms.

Ultra-sensitive label-free electrochemical detection of the acute leukaemia gene Pax-5a based on enzyme-assisted cycle amplification

Accurate and sensitive detection of the Pax-5a gene is of great importance in the early diagnosis and prognosis of acute leukaemia. Herein, a label-free electrochemical sensing system was proposed for the detection of the acute leukaemia Pax-5a gene based on enzyme-assisted signal amplification to generate abundant G-quadruplex/hemin DNAzyme. The presence of Pax-5a can open the hairpin probe (HP), which acts as a template. Under the action of the restriction enzymes Nt.BbvCI and Klenow fragment polymerase, the target gene Pax-5a is cycled to open the HP; On the other hand, a large number of G-quadruplex sequences are produced. The resulting G-quadruplex sequence is capable of forming the G-quadruplex/hemin complex on the surface of the electrode in the presence of hemin. The ultrasensitive label-free electrochemical detection of Pax-5a can be realized via the G-quadruplex/hemin complex-catalysed reduction of H2O2, and the detection limit was estimated to be as low as 4.6 fM. In addition, the biosensor has good specificity and stability, and also has excellent detection capabilities in a complex substrate environment. Therefore, the sensor shows great potential in bioanalysis and clinical diagnosis.

A label-free colorimetric detection of microRNA via G-quadruplex-based signal quenching strategy

Signal amplification strategy have been the intensive direction for highly-sensitive microRNA (miRNA) detection, however, it is worth noting that signal quenching approach is rarely realized. Inspired by the unwinding of G-quadruplex with Klenow Fragment polymerase (KF), a simple and label-free signal quenching system for detection of miRNAs was developed. In the presence of target miRNA, miRNA was used as a primer to promote KF activity resulting in the disruption of G-quadruplex structure of probe and the biological catalysis inactivation of DNAzyme formed by G-quadruplex structure and hemin. As a result, the probe would be transformed from G-quadruplex to duplex and the obvious differential signal could be observed in most cases even with naked eyes. With the significant signal decreasing, the target miRNA can be rapidly analyzed by UV–vis spectra with a considerably low detection limit (4.5 nM). This method exhibits excellent selectivity and anti-interference ability by discriminating base-mismatched and other miRNA families from target miRNA. Meanwhile, a good recovery (90.7%∼102.4%) in 5% human serum was obtained and this strategy verified a high expression level of miR21 in total RNA of MCF-7 compared with 293T and HeLa. It implies that this assay is of great potential to be applied in biochemical research and clinical diagnosis.

Palindromic Fragment-Mediated Single-Chain Amplification: An Innovative Mode for Photoelectrochemical Bioassay.

This work reports a strategy for glutathione-loaded liposome-encoded magnetic beads initiated by palindromic fragment-mediated single-chain amplification (PFMSCA) for high-precision quantification of a low-abundance aminoglycoside antibiotic (kanamycin; Kana) by using In2O3-ZnIn2S4 (IO-ZIS) as a photoactive matrix. In this strategy, a Kana-recognition region, primer-like palindromic fragment, and polymerization/nicking template are reasonably integrated into one oligonucleotide (hairpin HP1) for target recognition, magnetic separation, and target amplification. Upon target Kana introduction, the Kana-aptamer region in HP1 specifically recognizes the Kana and triggers the palindromic tails intramolecular self-hybridization, amplifying a large number of short fragments in the presence of Klenow fragment polymerase and Nt.BbvCI. The as-generated nick fragments act as a linker to introduce the free hairpin HP2-functionalized glutathione-loaded liposomes (HP2-GLL) onto the surface of the hairpin HP3-modified magnetic beads (HP3-MB), constructing liposome-encoded magnetic beads (HP3-MB-nick-HP2-GLL). Following magnetic separation, the detached glutathione-loaded liposomes (GLL) are lysed by treatment with 1% Triton X-100 to release the glutathione within it, which were then detected as an amplified photocurrent at the IO-ZIS-based photoelectrode. Importantly, this method can be readily carried out by using one oligonucleotide to achieve an exponential amplification effect and open new opportunities for advanced development of robust biodetection systems.

Triplex DNA: A new platform for polymerase chain reaction-based biosensor.

Non - specific PCR amplification and DNA contamination usually accompany with PCR process, to overcome these problems, here we establish a sensor for thrombin by sequence - specific recognition of the PCR product with molecular beacon through triplex formation. Probe A and probe B were designed for the sensor, upon addition of thrombin, two probes hybridized to each other and the probe B was extended in the presence of Klenow Fragment polymerase and dNTPs. The PCR amplification occurred with further addition of Taq DNA Polymerase and two primers, the PCR product was recognized by molecular beacon through triplex formation. The fluorescence intensity increased with the logarithm of the concentration of thrombin over the range from 1.0 × 10−12 M to 1.0 × 10−7 M, with a detection limit of 261 fM. Moreover, the effect of DNA contamination and non - specific amplification could be ignored completely in the proposed strategy.

Molecular Dynamics Study of the Opening and Closing Mechanisms for DNA Polymerase I

The quick and accurate replication of genetic information (DNA) is critical to all life. DNA polymerase, a key enzyme for DNA replication in organisms, catalyzes the reaction that extends the primer strand of DNA during replication. Before catalysis, DNA polymerase has the daunting task of matching a template nucleic acid base with its corresponding Watson‐Crick base (dNTP) that must be added to the primer strand. The enzyme must have high specificity in order to distinguish one correct base for incorporation from three incorrect bases. Crystal structures of DNA polymerase have shown the enzyme in an “open” conformation in the absence of a dNTP in the active site and “closed” when a proper Watson‐Crick base pair is matched. Although crystal structures have given insight into these conformations of DNA polymerase in these three states, traditional biochemistry experimental techniques are unable to capture the details of the transition between these states and how the polymerase traverses the potential energy surface from open to closed and back again. In this study, we have utilized recent computational advances to simulate the opening and closing of the fingers domain starting from the of Bacillus stearothermophilus DNA polymerase Klenow fragment using dynamics on the microsecond time scale. We have fully characterized both processes, and determined the key events and movements critical to these large‐scale conformational changes. These simulations aid in the elucidation of the structural changes of DNA polymerase on an atomistic level not currently available with experimental measures.

Cleaved DNAzyme substrate induced enzymatic cascade for the exponential amplified analysis of l-histidine

A novel strategy of cleaved DNAzyme substrate induced enzymatic cascade has been devised for the exponential amplified detection of l-histidine. The enzyme strand carries out hydrolytic cleavage of the substrate strand in the presence of l-histidine. The cleaved DNAzyme substrates introduce the polymerase/endonuclease reaction cycles as primers. The l-histidine acts as the activator for enzymatic cascade amplification generating a distinguishable fluorescence enhancement. A good nonlinear correlation (R=0.9994) between fluorescence intensity and the logarithm of the l-histidine concentration is obtained over the range from 50nM to 1.0mM. The detection limit was estimated as 30nM. This efficient amplification of the fluorescence signal is attributed to the l-histidine induced cooperation of Klenow Fragment polymerase (exo−) and Nb.BbvCI endonuclease reaction. The activation of such enzymatic cascades through analyte-DNAzyme interactions has a substantial impact on the development of exponential amplified DNAzyme sensors.

Analysis of hepatitis e virus-like sequence in chimpanzee.

Dear Editor, Hepatitis E virus (HEV), a member of the genus Hepevirus, is a non-enveloped virus with a positive stranded RNA genome that is approximately 7.2 kb in length (1). It has been hypothesized that zoonosis is involved in the transmission of HEV (2, 3). Hepatitis E virus antibodies or genes have been reported to exist in many species of mammals, including monkeys (4, 5). Recently, divergent HEV strains has been discovered in different animals, including rats (6), mouses (7), and rabbits (8), which suggests that more animal species could be the reservoir of HEV. In the present study, we analyzed a HEV-like sequence, which was found by chance during the discovery of RNA virus in fecal samples of Chimpanzee from a zoo in China. Briefly, we extracted total RNA from the fecal suspension and performed reverse transcription using a primer containing a fixed sequence followed by a randomized octomer at the 3′ end. A single round of DNA synthesis was then performed using Klenow fragment polymerase. Twenty cycles of PCR amplification of nucleic acids was then performed using primers consisting of fixed portions of the random primers. Then the PCR products were purified, cloned into T-vector and sequenced. The resulting sequences were searched in GenBank using BLASTx. Searching results showed that one 685 bp sequence had the highest sequence homology with HEV, sharing 45-58% sequence identities. Sequence analysis revealed that the putative amino acid sequence of this fragment included the whole RdRp domain, which contained 157 amino acids. Due to the high divergence of the sequence, multiple attempts to acquire longer sequences of this virus failed. In order to investigate whether this sequence is prevalent in the Chimpanzee population, a set of primers were designed according to the 685 bp sequence in the present study to perform PCR screening in 24 fecal samples collected from Chimpanzees at the same zoo and 13 fecal samples from another zoo in China. The primers were Chev1 [5’-TGTCCTCATGTCTGTCAGG-3’] and Chev2 [5’-AATCACATCTACCAACAGC-3’] for the first round of PCR, and Chev3 [5’-TGCCACGGTCCACCGATCG-3’] and Chev4 [5’-ATAGAACCACCGGCGTTG-3’] for the second round. This set of nested primers was designed to amplify a 154-nt segment. Our PCR screening results indicated that seven (29.2%) of the 24 fecal samples from the same zoo were positive for this HEV-like sequence, while none of the 13 samples from the other zoo were positive, which suggests that this virus strain was highly prevalent in the Chimpanzee population at the studied zoo. The seven positive samples were cloned and sequenced; results indicated that they shared > 99% identity over nucleotide level, suggesting they belonged to the same virus strains. In order to further identify the genetic relationship between the sequences of this study and other known HEV strains, we performed a phylogenetic analysis based on the predicted amino acid sequences in the current study and those related sequences retrieved from GenBank. The HEV sequences included those from well-known HEV genotypes 1-4 (from human or pig), rat HEV, mouse HEV, rabbit HEV, and avian HEV. The other four related virus sequences were also added as outgroups, including hepatitis A virus, sapovirus, rabbit vesivirus and rabbit hemorrhagic disease virus. Briefly, amino acid sequences were aligned using Clusta lW v2.0. Phylogenetic analysis was constructed using the Mega 5 software (http://www.megasoftware.net/). GenBank accession numbers of the sequences used as references in this analysis are shown in Figure 1. The sequences determined in the current study were deposited in GenBank; strain name being Nhev-Cb1 and the accession number {type:entrez-nucleotide,attrs:{text:KM407530,term_id:676260547,term_text:KM407530}}KM407530. Our phylogenetic analysis indicated that Nhev-Cb1 clustered with the other HEV sequences, lying in a deep branch with high bootstrap value of 100 (Figure 1). Over the amino acid sequence level, Nhev-Cb1 shared 35-39% sequence identity with the other HEV species, and 29-32% sequence identities with the four reference sequences. Our phylogenetic results suggested that the virus in the present study was a novel type of HEV. Although the antibodies to HEV and human origin HEV genes were discovered in non-human primates (4, 5, 9), these animals are not considered as the natural reservoirs for HEV. In the present study, HEV-like sequences were detected in seven (29.2%) of 24 Chimpanzees at the studied zoo, suggesting that if the sequences are from real viral particles, the virus may be a new type of HEV using non-human primate as its natural host. Figure 1. Phylogenetic Analysis of the HEV-Like Sequence in the Present Study and the Reference Sequences from GenBank

Amplified Chemiluminescence Detection of DNA by Strand-Displacement Polymerization Target Recycling and G-quadruplexes/Hemin DNAzyme Catalysis

A new strategy based on strand-displacement polymerization target recycling and G-quadruplex DNAzyme catalysis was developed for amplified chemiluminescence detection of DNA. The amplified detection was achieved by using the system consisted of hairpin DNA probe, G-rich DNA, primer, and polymerase Klenow fragment exo−. When the target DNA was introduced the system, the hairpin structure of DNA probe was opened by the hybridizing of target DNA with its complementary sequence, the primer hybridized then with DNA probe and initiated polymerase-aided strand-displacement polymerization reaction, resulting in the release of target DNA and G-rich DNA. The released target DNA again hybridizes with another DNA probe to trigger the next polymerase-aided strand-displacement polymerization, generating large numbers of G-rich DNA. The G-rich DNA assembles with hemin to form the G-quadruplexes/hemin DNAzyme, which can catalyze oxidation of luminol by H2O2, generating chemiluminescence. This unique amplifying strategy gives a detection limit down to 2.5 pM, which is at least two to three orders of magnitude lower than that of unamplified DNA detection methods.

A quantitative fluorescence-based steady-state assay of DNA polymerase.

Fluorescent dyes that bind DNA have been demonstrated as a useful alternative to radionucleotides for the quantification of DNA and the in vitro measurement of the activity of DNA polymerases and nucleases. However, this approach is generally used in a semi-quantitative way to determine relative rates of reaction. In this report, we demonstrate a method for the simultaneous quantification of DNA in both its single-strand and double-strand forms using the dye PicoGreen. This approach is used in a steady-state assay of DNA polymerase Klenow fragment exo(−), where we determine kcat and Km values for the DNA polymerase that are in excellent agreement with literature values.

Isothermal Strand-Displacement Polymerase Reaction for Visual Detection of the Southeast Asian–Type Deletion of α-Thalassemia

A rapid and reliable screening test for thalassemia carrier couples is the most effective strategy to decrease the risk of conceiving fetuses with severe thalassemia. We present an approach based on the isothermal strand-displacement polymerase reaction and the use of a lateral flow strip for the visual detection of an α-thalassemia Southeast Asian-type deletion. This assay was used to evaluate 86 clinical samples (72 cases of Southeast Asian-type deletions and 14 cases of other types of thalassemia), and the results obtained were 100% consistent with those obtained using conventional gap-PCR. The approach thus provides a simple, sensitive, rapid, and cost-effective method for the diagnosis of thalassemia genotypes.

Single Molecule Dynamic Transduction by Carbon Nanotube Circuits

Nanoscale electronic devices like field-effect transistors (FETs) have long promised to provide sensitive, label-free detection of biomolecules. In particular, single-walled carbon nanotubes (SWNTs) have the requisite sensitivity to detect single molecule events, and have sufficient bandwidth to directly monitor single molecule dynamics in real time. Recent measurements have demonstrated this premise by monitoring the dynamic, single-molecule processivity of three different enzymes: lysozyme, protein kinase A, and the Klenow Fragment of polymerase I. Initial successes in each case indicated the generality and attractiveness of SWNT FETs as a new tool to complement other single molecule techniques. However, further generalization of the SWNT FET technique demands reliable design rules that can predict the success and applicability of these devices. Here, we address this need by focusing on the transduction mechanisms that link enzyme processivity to electrical signal generation in a SWNT FET. Using ten different lysozyme variants synthesized by mutagenesis, we systematically dissect the enzyme-SWNT interaction in order to understand this transduction. The data prove that mechanical displacements of charged functionalities near the SWNT attachment site are the primary sources of transduction, and that the resulting devices are sensitive enough to track the motions of just one charged amino acid. The purposeful incorporation of a charged group at a particular location allows the device to be designed to have sensitivity to particular chemical activities or allosterically-driven mechanical motions. The findings provide rules for the creation of similarly effective nanocircuits using a wide range of enzymes or proteins.

A graphene oxide platform for the assay of DNA 3'-phosphatases and their inhibitors based on hairpin primer and polymerase elongation.

We have developed a label-free, simple and highly sensitive hairpin fluorescent biosensor for the assay of DNA 3'-phosphatases and their inhibitors utilizing a graphene oxide (GO) platform. In this assay, we designed a hairpin primer (HP) with a 3'-phosphoryl end that served as the substrate for DNA 3'-phosphatases. Once the phosphorylated HP was hydrolyzed by DNA 3'-phosphatases, the resulting HP with a 3'-hydroxyl end was immediately elongated to form a long double-strand product by Klenow fragment polymerase (KF polymerase). With SYBR green I (SG) selective staining of the double-helix DNA, a very high fluorescence enhancement was achieved. Furthermore, GO was introduced to quench the fluorescence of the HP without polymerase elongation, thereby further increasing the signal-to-background ratio. The proposed method is simple and convenient, yet still exhibits high sensitivity and selectivity. This method has been successfully applied to detecting the activity of two typical 3'-phosphatases, T4 polynucleotide kinase phosphatase (PNKP) and shrimp alkaline phosphatase (SAP). The effect of their inhibitors has also been investigated. The results revealed that the method allowed a sensitive quantitative assay of T4 PNKP and SAP, with detection limits of 0.07 U mL-1 and 0.003 U mL-1, respectively. The proposed method is anticipated to find applications in the study of DNA damage repair mechanisms.

KF polymerase-based fluorescence aptasensor for the label-free adenosine detection

We have developed a simple, inexpensive, and label-free method for the selective detection of adenosine. Klenow fragment polymerase (KF polymerase) is a commonly-used 5' to 3' DNA polymerase, it also has 3' to 5' exonuclease activity that can digest single-stranded DNA. An adenosine binding DNA aptamer was employed, the aptamer was split into two pieces of single-stranded DNA (aptamer-A1 + aptamer-A2). Without the addition of adenosine, aptamer-A1 and aptamer-A2 existed as single-stranded DNA which could be efficiently degraded by the exonuclease activity of KF polymerase. Much reduced background fluorescence was obtained when SYBR Green dye was added. However, in the presence of adenosine, aptamer-A1 and aptamer-A2 bound to adenosine, and hybridization of the complementary sequences resulted in the formation of a duplex DNA structure, which could initiate DNA polymerization. The addition of SYBR Green dye resulted in a very high fluorescence enhancement, which could be used for the quantification of adenosine.

Electrochemical aptameric sensor based on the Klenow fragment polymerase reaction for cocaine detection

An electrochemical aptasensor based on Klenow fragment (KF) polymerase reaction that combines the aggregation of ferrocene-functionalized oligonucleotide has been developed successfully for cocaine detection. In the presence of cocaine, the recognition probe changed its hairpin conformation into the tripartite complex. The aptamer-cocaine complex gave a 3′-single-stranded tail sequence complementary to the surface-tethered capture probe. In KF polymerase reaction, the recognition probe served as a template for the extension of a capture probe. It requires a sample volume of 2 μL and is complete within 1 h. The ferrocene-appended oligonucleotide incorporated into the newly synthesized complementary probe leads to an electrochemical response. This sensitive detection of cocaine is due to a very low background signal and large signal enhancement up to 9-fold upon addition of analyte. It permits detection of as low as 200 μM cocaine. The simple and isothermal procedure does not require thermal cycling or special laboratory conditions, which makes it adaptable to low-cost and robust biosensing.

Whole Genome Amplification by T7-Based Linear Amplification of DNA (TLAD): II. Second-Strand Synthesis and In Vitro Transcription

INTRODUCTIONT7-based linear amplification of DNA (TLAD) uses a linear amplification approach based on in vitro transcription (IVT) of template DNA by RNA polymerase from the T7 phage. TLAD was designed primarily for use with the ChIP-chip method (whereby DNA recovered from chromatin immunoprecipitation [ChIP] of cell lysate is used for subsequent analysis on DNA microarrays) and requires nanogram quantities of dsDNA to generate microgram amounts of amplified RNA. Briefly, the strategy is to add a 3' conserved end to the template dsDNA, using terminal deoxynucleotidyl transferase (TdT) tailing, which permits the addition of a T7 promoter sequence in the subsequent second-strand synthesis step, described here. At this stage, the strand-displacement activity of the Klenow fragment polymerase separates the two strands of the template DNA, after which the enzyme performs fill-in 5' → 3' polymerization. Its 3' → 5' exonuclease activity may also remove the 3' overhanging poly(dT) tails, although the efficiency of this activity will vary based on the length of the poly(dT) tail. IVT can then use this newly appended T7 promoter. Because the T7-based IVT proceeds as an isothermal reaction, it linearly amplifies the template DNA, producing antisense RNA (aRNA) (i.e., each strand of RNA produced is antisense to the original template strand). Since both strands are amplified, this distinction is usually not important and is affected only by the location of the T7 promoter and poly(A) tract on the aRNA.

Nuclear Receptor CAR Requires Early Growth Response 1 to Activate the Human Cytochrome P450 2B6 Gene

The nuclear receptor CAR (constitutive active/androstane receptor) is a drug-sensing transcription factor, regulating the hepatic genes that encode various drug-metabolizing enzymes. We have now characterized the novel regulatory mechanism by which the signal molecule EGR1 (early growth response 1) determines CAR-mediated activation of the human CYP2B6 (cytochrome P450 2B6) gene. The CYP2B6 enzyme metabolizes commonly used therapeutics and also activates pro-drugs. The CAR directly binds to the distal enhancer element of the CYP2B6 promoter, which is essential in converging to its drug-sensing function onto promoter activity. However, this binding alone is not sufficient to activate the CYP2B6 promoter; the promoter requires EGR1 to enable CAR to activate the CYP2B6 promoter. Upon stimulation by protein kinase C, EGR1 directly binds to the proximal promoter and coordinates the nearby HNF4alpha (hepatocyte-enriched nuclear factor 4alpha) with CAR at the distal enhancer element to activate the promoter. Thus, synergy of drug activation and the stimulation of cellular signal are necessary for CAR to activate the CYP2B6 gene.

Conserved ETS Domain Arginines Mediate DNA Binding, Nuclear Localization, and a Novel Mode of bZIP Interaction

The DNA-binding ETS transcription factor Spi-1/PU.1 is of central importance in determining the myeloid-erythroid developmental switch and is required for monocyte and osteoclast differentiation. Many monocyte genes are dependent upon this factor, including the gene that codes for interleukin-1beta. It has long been known that the conserved ETS DNA-binding domain of Spi-1/PU.1 functionally cooperates via direct association with a diverse collection of DNA-binding proteins, including members of the basic leucine zipper domain (bZIP) family. However, the molecular basis for this interaction has long been elusive. Using a combination of approaches, we have mapped a single residue on the surface of the ETS domain critical for protein tethering by the C/EBPbeta carboxyl-terminal bZIP domain. This residue is also important for nuclear localization and DNA binding. In addition, dependence upon the leucine zipper suggests a novel mode for both protein-DNA interaction and functional cooperativity.