Nick Region Research Articles

Signal-amplified detection of biomarker transcription factors with improved accuracy based on T4 DNA ligase protection and rolling circle amplification

Transcription factors (TFs) are emerging tumor biomarkers that regulate gene expression. The detection of TFs depends on the transduction of TF-DNA binding to the nucleic acid signal, which mostly relies on exonuclease III (Exo III) protection assays. However, the processive digestion of Exo III usually causes undesired dissociation of the TF-DNA complex, resulting in low assay accuracy. We proposed a strategy, named T4 DNA ligase protection (T4LiP), to transduce the binding signal and then coupled it with rolling circle amplification (RCA) to fluorescently detect TFs. TFs bound to the nick region of dsDNA can prevent the association of T4 DNA ligase with the nick, thus inhibiting the ligation. Because of the mild activity of T4 DNA ligase, negligible dissociation of the TFs from dsDNA was observed. Using this to our advantage, we transduced the TF-DNA binding signal to generate circular DNA and then loaded it with RCA to sensitively detect p50 protein, yielding a detection limit of 0.5 pM. By sequence engineering, the detection of multiple TFs including MITF, p53, AP-1, and c-Myc with low picomolar sensitivities was accomplished. This is the first report of ligase-assisted transduction of TF-DNA binding, which shows high stability and versatility and holds the potential to improve the performance of TF assays.

Validation of a hepatitis B virus (HBV) Next Generation Sequencing (NGS) pipeline at Barts Health NHS Trust

At Barts Health NHS Trust, historically HBV genotyping and resistance testing is performed using nested PCR followed by Sanger sequencing. With the development of NGS assays for HIV and HCV, in order to harmonise workflows an NGS HBV pipeline was developed. Primers were designed for the whole genome amplification of HBV. Plasma samples were extracted for nucleic acid using a Qiagen Qiasymphony. Whole genomes of HBV were amplified in a single round PCR and quantified using a Qubit fluorometer. Libraries were prepared using the Nextera XT library preparation kit and sequencing was performed on an Illumina Miseq. Sequencing reads were assembled into consensus sequences using a Linex-based pipeline. Resistances and genotypes were determined using the HBV Grade website. Minority variants were variants <20 % of the overall population. Pan-genotypic PCR primers were designed in the nick region of the partially double stranded HBV genome. In-silico analysis allowed primers to be designed to bind multiple genotypes. Sensitivity analysis of primers was investigated for the most common circulating genotypes. A panel of HBV positive and external quality assurance samples showed good comparability to Sanger sequencing results. Inter- and intra-assay variability showed the assay was robust and fit for purpose. No resistance associated minority variants were observed. The development an NGS pipeline for analysis of HBV allowed the harmonisation of diagnostic pathways within the virology sequencing laboratory. Introduction of this test would allow data to be gathered to assess the importance of minority variants in HBV infection.

Site-specific Relaxase Activity of a VirD2-like Protein Encoded within the tfs4 Genomic Island of Helicobacter pylori

Four different type IV secretion systems are variously represented in the genomes of different Helicobacter pylori strains. Two of these, encoded by tfs3 and tfs4 gene clusters are contained within self-transmissible genomic islands. Although chromosomal excision of tfs4 circular intermediates is reported to be dependent upon the function of a tfs4-encoded XerD tyrosine-like recombinase, other factors required for transfer to a recipient cell have not been demonstrated. Here, we characterize the functional activity of a putative tfs4-encoded VirD2-like relaxase protein. Tfs4 VirD2 was purified as a fusion to maltose-binding protein and demonstrated to bind and nick both supercoiled duplex DNA and oligonucleotides in vitro in a manner dependent upon the presence of Mg(2+) but independently of any auxiliary proteins. Unusually, concentration-dependent nicking of duplex DNA appeared to require only transient protein-DNA interaction. Although phylogenetically distinct from established relaxase families, site-specific cleavage of oligonucleotides by Tfs4 VirD2 required the nick region sequence 5'-ATCCTG-3' common to transfer origins (oriT) recognized by MOBP conjugative relaxases. Cleavage resulted in covalent attachment of MBP-VirD2 to the 5'-cleaved end, consistent with conventional relaxase activity. Identification of an oriT-like sequence upstream of tfs4 virD2 and demonstration of VirD2 protein-protein interaction with a putative VirC1 relaxosome component indicate that transfer initiation of the tfs4 genomic island is analogous to mechanisms underlying mobilization of other integrated mobile elements, such as integrating conjugative elements, requiring site-specific targeting of relaxase activity to a cognate oriT sequence.

Mapping of the nick site on conjugative plasmid pVT745 by interrupted mating

Conjugal transfer of plasmid DNA initiates and terminates at a specific non-coding site called the origin of transfer ( oriT). Previous analysis of conjugative plasmid pVT745 from Aggregatibacter actinomycetemcomitans suggested that oriT was located adjacent to the operon responsible for initiation of ssDNA transfer. The location of oriT was confirmed by assaying both subclones of the region as well as a pVT745 deletion derivative for mobilization. The precise DNA nick site ( nic) and polarity of DNA transfer were identified by use of interrupted mating assays, a technique originally used for the mapping of bacterial chromosomes. Nucleotide sequence analysis revealed that the pVT745-specific nick region was similar to the consensus nick sequence of the IncP family albeit the actual cleavage site differed. Functionality of nic was confirmed by point mutations.

Structural Basis of the Role of the NikA Ribbon-Helix-Helix Domain in Initiating Bacterial Conjugation

Conjugation is a fundamental process for the rapid evolution of bacteria, enabling them, for example, to adapt to various environmental conditions or to acquire multi-drug resistance. NikA is one of the relaxosomal proteins that initiate the intercellular transfer of the R64 conjugative plasmid with the P-type origin of transfer, oriT. The three-dimensional structure of the N-terminal 51 residue fragment of NikA, NikA(1–51), which binds to the 17-bp repeat A sequence in R64 oriT, was determined by NMR to be a homodimer composed of two identical ribbon-helix-helix (RHH) domains, which are commonly found in transcriptional repressors. The structure determination of NikA(1–51) was achieved using automated NOE assignment with CYANA, without measuring filtered NOESY experiments to distinguish between the intra- and intermolecular NOEs, and without any a priori assumption on the tertiary or quaternary structure of the protein. Mutational experiments revealed that the DNA-binding region of the NikA(1–51) dimer is an anti-parallel β-sheet composed of one β-strand from each of the N-terminal ends of the two domains. Various biochemical experiments have indicated that the full length NikA(1–109) exists as a homotetramer formed through an α-helical domain at the C-terminus, and that the anti-parallel β-sheets of both dimeric domains bind to two homologous 5 bp internal repeats within repeat A. As a tetramer, the full length NikA(1–109) showed higher affinity to repeat A and bent the oriT duplex more strongly than NikA(1–51) did. Many RHH proteins are involved in specific DNA recognition and in protein–protein interactions. The discovery of the RHH fold in NikA suggests that NikA binds to oriT and interacts with the relaxase, NikB, which is unable to bind to the nick region in oriT without NikA.

CD and Melting Curves Structural Studies of the Tandem DNA Complex Formed with Oligonucleotides Carrying Photoactive and Sensitizing Groups in the Nick Region

Photoactive derivatives of oligonucleotides are widely used as affinity reagents for the study of structures and functions of nucleic acids and proteins. Between them the binary reagents are the more attractive in the last time. They represent the tandem of two oligonucleotide derivatives complementary to a target sequence and carrying photoactive and sensitizing groups. The efficiency of target modification in this case depends on the mutual arrangement in the nick region of photoactive and sensitizing groups, attached to the oligonucleotides. The use of binary reagents in affinity modification permits to reach the high selectivity of the process. In this work we report our studies on the thermodynamic and structural peculiarities of complementary tandem complex between DNA target and binary oligonucleotide reagent. The complex consisted of the target d(TTGAAGGGGACCGC) and two 7-mer oligonucleotide conjugates, one of which was modified on its 3′-phosphate with a photoreactive p-azidote-trafluorobenzaldehydehydrazone-group, and the other one was linked through its 5′-phosphate to a sensitizing perylene-group. Optical melting curves and thermal changes in circular dichroism (CD) spectra were detected for all possible oligonucleotide and/or conjugate combinations. In addition, molecular modeling simulation of the complex structure was carried out. It was found that CD spectra did not show serious changes in the B-helix structure of the duplex. The interaction between perylene- and azido-groups at the oligonucleotide junction led to considerable increase in duplex stability. CD and molecular modeling data clearly indicated that perylene-group interacted with the duplex in an intercalative manner, but azido-group located on the side of DNA chain minor groove.

Oligonucleotide Tandem Complex Stabilization in the Presence of Photoactivated and Sensitizing Groups in the Nick Region

Oligonucleotide Tandem Complex Stabilization in the Presence of Photoactivated and Sensitizing Groups in the Nick Region

Initiation and termination of DNA transfer during conjugation of IncI1 plasmid R64: roles of two sets of inverted repeat sequences within oriT in termination of R64 transfer.

Intercellular transfer of plasmid DNA during bacterial conjugation initiates and terminates at a specific origin of transfer, oriT. We have investigated the oriT structure of conjugative plasmid R64 with regard to the initiation and termination of DNA transfer. Using recombinant plasmids containing two tandemly repeated R64 oriT sequences with or without mutations, the subregions required for initiation and termination were determined by examining conjugation-mediated deletion between the repeated oriTs. The oriT subregion required for initiation was found to be identical to the 44-bp oriT core sequence consisting of two units, the conserved nick region sequence and the 17-bp repeat A sequence, that are recognized by R64 relaxosome proteins NikB and NikA, respectively. In contrast, the nick region sequence and two sets of inverted repeat sequences within the 92-bp minimal oriT sequence were required for efficient termination. Mutant repeat A sequences lacking NikA-binding ability were found to be sufficient for termination, suggesting that the inverted repeat structures are involved in the termination process. A duplication of the DNA segment between the repeated oriTs was also found after mobilization of the plasmid carrying initiation-deficient but termination-proficient oriT and initiation-proficient but termination-deficient oriT, suggesting that the 3' terminus of the transferred strand is elongated by rolling-circle-DNA synthesis.

Refined High-Field NMR Solution Structure of a Binary-Addressed Pyrene/Perfluoro-Azide Complementary DNA Oligonucleotide System Shows Extensive Distortion in the Central Nick Region

The structural analysis of the photoactivated binary system of complementary-addressing nucleic acid sequences (1:2:3) by high-resolution NMR spectroscopy and restrained molecular dynamics is reported. The binary system comprised a 12 base-pair target DNA sequence, pdGTATCAGTTTCT (1), and two hexanucleotides, (dAGAAACp-L-Az (2) and PyrpdTGATAC (3)), complementary to neighbouring sites in the target DNA. Oligonucleotide (2) is conjugated with a p-azidotetrafluo'robenzyl group (Az) via a linker group (L), and the other oligonucleotide (3) is equipped with the photosensitizing pyrenyl-l-methylamino group (Pyr). We now extend the structural analysis of 1:2:3, which was previously based on qualitative 2D 1H-NMR data and thermodynamic analysis of complex formation from UV-visible thermal denaturation experiments. In the current work structural refinement was performed by separate molecular dynamics runs for six different starting structures based on 318 protonproton distance-range constraints, evaluated from the 1H-NOESY spectrum (Tmix=200rns, 600MHz) using complete relaxation matrix analysis (NMR/TRIAD/MARDIGRAS). Additional Watson-Crick hydrogen bond restraints were included in the calculations based on the detected signals from the exchangeable protons, using REFOPT(NY) methods. The final averaged structure obtained from the six refined co-ordinate sets showed a considerable degree of axis bend (62.5°) with the bending point in the middle of the duplex in the region of the backbone nick between the two short oligonucleotides. The complex behaves dynamically as the equivalent of two short B-DNA-like duplexes displaying a hinge-like flexing at their junction. In all final structures the Pyr function location was very restricted, the aromatic group lying in the duplex minor groove near residues 4T, 5C and 20T. In contrast, the location of the perfluoroazido group was different in all the final structures, indicating the high flexibility of this group in the duplex. The only feature common to all six final azido group orientations was the outside location on the side of the major groove. The distance between the Pyr and Az groups varied from 11Å to 24Å for the six final structures (17Å, final average structure). The dynamics of duplex denaturation for 1:2:3 was probed by monitoring the temperature-induced NMR line broadening of the imino protons in a ID variable temperature NMR experiment. The melting of 1:2:3 starts both from the ends and from the middle part of the duplex at the backbone break between the two short oligonucleotides reflecting the destabilisation of the pyrene-arylazido nick region in the duplex.

Mutational analysis of the R64 oriT region: requirement for precise location of the NikA-binding sequence.

Conjugative DNA transfer of IncI1 plasmid R64 is initiated by the introduction of a site- and strand-specific nick into the origin of transfer (oriT). In R64 oriT, 17-bp (repeat A and B) and 8-bp inverted-repeat sequences with mismatches are located 8 bp away from the nick site. The nicking is mediated by R64 NikA and NikB proteins. To analyze the functional organization of the R64 oriT region, various deletion, insertion, and substitution mutations were introduced into a 92-bp minimal R64 oriT sequence and their effects on oriT function were investigated. This detailed analysis confirms our previous prediction that the R64 oriT region consists of an oriT core sequence and additional sequences necessary for full oriT activity. The oriT core sequence consists of the repeat A sequence, which is recognized by R64 NikA protein, and the nick region sequence, which is conserved among various origins of transfer and is most probably recognized by NikB protein. The oriT core sequence is sufficient for NikAB-mediated oriT-specific nicking. Furthermore, it was shown that the repeat A sequence is essential for localization to a precise position relative to the nick site for oriT function. This seems to be required for the formation of a functional ternary complex consisting of NikA and NikB proteins and oriT DNA. The repeat B sequence and 8-bp inverted repeat sequences are suggested to be required for the termination of DNA transfer.

Functional Characterization of the P2 A Initiator Protein and Its DNA Cleavage Site

The A protein of bacteriophage P2 initiates DNA replication by a single-stranded cut at the origin, and the DNA replication proceeds unidirectionally by a modified rolling circle type of replication. The P2 A protein belongs to a family of proteins involved in the initiation of rolling circle DNA replication, and the prototype for this family is the well-characterized A protein of phage φX174. One of the common motifs of this family contains two conserved tyrosine residues, which have been shown to be able to alternate in catalyzing the cleavage as well as joining reactions in the φX174 A protein. We investigated the role of the conserved tyrosine residues in P2 A protein byin vitromutagenesis. Only one of the two conserved tyrosine residues was found to be involved in the cleavage reaction. The tyrosine residue dispensable for cleavage and ligation is, however, required at some other stage of the P2 growth cycle, since viable recombinants containing this mutation could not be obtained. The sequence requirements for cleavage of the target site were analyzed with a set of oligonucleotides having single base alterations in the nick region, and the results indicate that only five core nucleotides need to be conserved for efficient cleavage.

Site-specific cleavage and joining of single-stranded DNA by VirD2 protein of Agrobacterium tumefaciens Ti plasmids: analogy to bacterial conjugation.

As an early stage of plant transformation by Agrobacterium tumefaciens, the Ti plasmid is nicked at the border sequences that delimit the T-DNA. Cleavage results in covalent attachment of VirD2 to the 5' terminal of the nicked strand by a process resembling initiation of DNA transfer that occurs in the donor cell during bacterial conjugation. We demonstrate that this cleavage can be reproduced in vitro: VirD2 protein, the border-cleaving enzyme, was overproduced and purified. Cleavage assays were performed with single-stranded oligodeoxyribonucleotides encompassing the Ti plasmid border region or the transfer origin's nick region of the conjugative plasmid RP4. VirD2 of pTiC58 cleaves both border- and nick region-containing oligonucleotides. However, the relaxase TraI of RP4 can cut only the cognate nick regions. The respective proteins remain covalently bound to the 5' end of the cleavage sites, leaving the 3' termini unmodified. VirD2-mediated oligonucleotide cleavage was demonstrated to be an equilibrium reaction that allows specific joining of cleavage products restoring border and nick regions, respectively. The possible role of VirD2 in T-DNA integration into the plant cell's genome is discussed in terms of less stringent target-sequence requirements.

Processes at the nick region link conjugation, T‐DNA transfer and rolling circle replication

Data from prokaryotic replicative and conjugative systems, which interrelate DNA processing events initiated by a site-specific nick, are reviewed. While the replicative systems have been established in accordance with the rolling circle replication model, the mechanism of conjugative replication has not been elucidated experimentally. We summarize data involving random point mutagenesis of the RK2 transfer origin (oriT), which yielded relaxation-deficient and transfer-deficient derivatives having mutations exclusively in a 10bp region defined as the nick region. Features of the RK2 (IncP) nick region, including the DNA sequence, nick site position, and 5' covalent attachment of the nicking protein, have striking parallels in other systems involving nicking and mobilization of single-stranded DNA from a supercoiled substrate. These other systems include T-DNA transfer occurring in Agrobacterium tumefaciens Ti plasmid-mediated tumorigenesis in plants, and the rolling circle replication of plasmids of Gram-positive bacteria and of phi X174-like bacteriophage. The structural and functional similarities suggest that IncP conjugative replication, originating at the oriT, and T-DNA transfer replication, originating at the T-DNA border, produce continuous strands via a rolling circle-type replication.

Indirect Evidence for Two Distinct Mechanisms of DNA Cleavage at the oriT of RP4 during Conjugation

Mobilization of a potential Campylobacter shuttle vector from Escherichia coli K-12 into C. hyointestinalis generated a small plasmid harboring a hybrid oriT nick region. This is presumed to have resulted from the cleavage and religation of the oriT of RP4 and the putative nick region of the shuttle vector. These data imply that two distinct DNA cleavage reactions occur at the oriT site of RP4 during conjugal transfer.

Relaxase (TraI) of IncP alpha plasmid RP4 catalyzes a site-specific cleaving-joining reaction of single-stranded DNA.

Conjugative DNA transfer of the self-transmissible broad-host-range plasmid RP4 is initiated by strand- and site-specific cleavage at the nick site (nic) of the transfer origin (oriT). Cleavage results in covalent attachment of the plasmid-encoded relaxase (TraI) to the 5'-terminal 2'-deoxycytidine residue at nic. We demonstrate that Tyr22 is the center of the catalytic site of TraI, mediating cleavage via formation of a phosphodiester between the DNA 5' phosphoryl and the aromatic hydroxyl group. The specificity of cleavage seen with form I oriT DNA was verified with short oligodeoxy-ribonucleotides embracing the nick region. The reaction requires TraI and Mg2+ but is independent of the relaxosome component TraJ. Cleavage produces one oligonucleotide fragment with a free 3' hydroxyl, the other part forms a covalent TraI-oligonucleotide adduct. Like nicking of form I oriT DNA, TraI-catalyzed oligonucleotide cleavage reaches an equilibrium when about 30% of the input TraI exists as a covalent protein-DNA complex. In the presence of two differently sized oligonucleotides, defined hybrid oligonucleotides are produced, demonstrating that TraI catalyzes recombination of two single strands at nic. This finding shows that TraI possesses cleaving-joining activity resembling that of a type I topoisomerase. Reactions are dependent on the sequence of the 3'-terminal 6 nucleotides adjacent to nic. Only certain base changes in a few positions are tolerated, whereas the sequence of the 5' terminal nucleotides apparently is irrelevant for recognition by TraI. The reactions described here further support the hypothesis that DNA transfer via conjugation involves a rolling circle-like mechanism which generates the immigrant single strand while DNA-bound TraI protein scans for the occurrence of a second cleavage site at the donor-recipient interface.

Characterization of the replication region of the small cryptic plasmid of Campylobacter hyointestinalis

The complete nucleotide sequence of a 2.5-kb cryptic plasmid from Campylobacter hyointestinalis was determined. Only one open reading frame (ORF), encoding a polypeptide of M r 39 667, designated RepA, could be identified within the sequence. This was confirmed by minicell analysis. Analysis of the region upstream from the ORF showed an A+T-rich region followed by four 19-bp direct repeats. Together, these features are characteristic of other replication origins ( ori(s)). The promoter sequence of the repA gene was identified by primer extension analysis and both the putative —10 and —35 regions were found to lie within two potential hairpin-loop structures. RepA showed marked amino acid sequence homology to a replication-initiation protein from the Neisseria gonorrhoeae plasmid, pFA3, and with other replication-initiation proteins over two conserved motifs. A putative partitioning ( par) locus was identified upstream from the ori and consisted of a perfect 9-bp inverted repeat and six putative DNA gyrase-binding sites. A putative mobilization origin ( oriT) region was identified. This featured a 19-bp imperfect inverted repeat adjacent to a sequence of 12 bp which showed strong homology to the consensus sequence of the ‘nick regions’ in a variety of oriTs of other plasmids.

Sequence identity in the nick regions of IncP plasmid transfer origins and T-DNA borders of Agrobacterium Ti plasmids

Sequence identity in the nick regions of IncP plasmid transfer origins and T-DNA borders of <i>Agrobacterium</i> Ti plasmids

Sequence identity in the nick regions of IncP plasmid transfer origins and T-DNA borders of Agrobacterium Ti plasmids.

The IncP antibiotic-resistance plasmids transfer to a broad range of bacterial species. The RK2 origin of DNA transfer (oriT) consists of a 250-base-pair segment including the single-stranded cleavage site (nic) needed to generate the DNA strand believed to be transferred. Deletion derivatives and a bank of hydroxylamine-generated oriT mutants were screened for loss of transferability. DNA regions flanking both sides of nic are required for optimal transfer of the oriT clone. Of the chemically induced mutants, critical base-pair changes that dramatically reduced transfer frequency were found in a 10-base-pair region adjacent to nic. Relaxation (nicking) assays performed with these point mutants using protein-DNA complexes reconstituted in vitro revealed a correlation between DNA nicking and transfer frequency. Base-pair changes within the proximal arm of an inverted repeat upstream from the nick site resulted in reduced binding of the essential transfer protein TraJ and correspondingly reduced transfer frequencies. The results support a model of relaxosome formation involving at least two essential proteins: TraI and TraJ. The nick region defined by the point mutants was located in a segment known to be nearly identical in the related plasmid R751. This sequence was also found to be highly conserved in both border junctions of the transfer DNA (T-DNA) of plant tumor-inducing plasmids of Agrobacterium tumefaciens, indicating a relationship between IncP-mediated broad-host-range bacterial conjugation and T-DNA transfer to plants.

Common sequence motifs in DNA relaxases and nick regions from a variety of DNA transfer systems.

Site and strand specific nicking at a transfer origin (oriT) is the central event in initiation of transfer DNA replication during bacterial conjugation. Recently, sequence identity in a DNA region adjacent to the nick sites of IncP plasmids RP4 and R751 and part of the border sequences of agrobacterial Ti and Ri plasmids has been demonstrated (Figure 1 and ref. 1). A search for this 'nick region' in a variety of transfer origins of other plasmids demonstrates its conservation in R64 (Incll) and pTFFC2, a mobilizable plasmid from Thiobacillus ferrooxidans (Figure 1, ref. 2). This finding is paralleled by occurrence of a common sequence motif in proteins (DNA-relaxases) encoded by the respective plasmids that interact with their cognate nick regions (Figure 2). Using the corresponding consensus sequence, a search in protein data bases (NBRF-Protein Release 27.0; Swiss-Prot Release 16.0) revealed that the 'relaxase motif is also occurring in potential DNA-relaxing proteins (Rlx) of mobilizable plasmids from S.aureus (Figure 2).

In situ nick translation of the fragile X region.

At the present time, the molecular nature of the fragile site at Xq27.3 is not well understood. To examine the sensitivity of this region to DNAase I, in situ nick translation was performed on metaphase chromosomes from a fragile X (fra(X] positive individual. In this technique DNAase I is used to nick regions of chromosomal DNA that are in "open" conformation. Biotinylated dUTP was incorporated by nick translation at these sites. The incorporation was identified by double antibody labeling and avidin-horseradish peroxidase staining. Spreads, which had been stained with this technique, were photographed and subsequently trypsin-Giemsa G banded (post-GTG banded) for chromosome identification. In 36 of 44 (82%) fra(X) positive male cells, the region distal to fra(X) (q27.3) was prominently stained in contrast to its light staining appearance in GTG preparations. The fragile site itself was outlined more clearly than can be achieved by GTG or homogeneous staining. When autosomal fragile sites were induced by the addition of 1.5 microM aphidicolin 17 hours prior to harvest, 24 of 27 (89%) fragile sites on the ends of autosomes were prominently stained in regions distal to the break. Because the fra(X) and autosomal fragile regions behaved similarly, this suggests that they have a similar conformation. Thus, while autosomal and Xq27.3 fragile sites are strongly induced by different means, the organization of these sites and the regions distal to them appear to be similar.