Unwinding of heterologous DNA by RecA protein during the search for homologous sequences

The search for homologous sequences promoted by RecA protein in vitro involves a presynaptic filament and naked duplex DNA, the multiple contacts of which produce nucleoprotein networks or coaggregates. The single-stranded DNA within the presynaptic filaments, however, is extended to an axial spacing 1.5 times that of B-form DNA. To investigate this paradoxical difference between the spacing of bases in the RecA presynaptic filament versus the target duplex DNA, we explored the effect of heterologous contacts on the conformation of DNA, and vice versa. In the presence of wheat germ topoisomerase I, RecA presynaptic filaments induced a rapid, limited reduction in the linking number of heterologous circular duplex DNA. This limited unwinding of heterologous duplex DNA, termed heterologous unwinding, was detected within 30 seconds and reached a steady state within a few minutes. Presynaptic filaments that were formed in the presence of ATP?S and separated from free RecA protein by gel filtration also generated a ladder of topoisomers upon incubation with relaxed duplex DNA and topoisomerase. The inhibition of heterologous contacts by 60 mImage -NaCl or 5 mImage -ADP resulted in a corresponding decrease in heterologous unwinding. In reciprocal fashion, the stability or number of heterologous contacts with presynaptic filaments was inversely related to the linking number of circular duplex DNA. These observations show that heterologous contacts with the presynaptic filament cause a limited unwinding of the duplex DNA, and conversely that the ability of the DNA to unwind stabilizes transient heterologous contacts.

DNA topoisomerase I from mycobacteria - A potential drug target

DNA topoisomerases are ubiquitous group of enzymes altering the topology of DNA by concerted breakage and rejoining of the phosphodiester backbone of DNA. The enzymes are classified based on the pattern of DNA cleavage. Type IA enzymes found in all bacteria nick the DNA and attach themselves covalently to the 5' side of the nick during the first transesterification reaction. Most of the information on this group of enzymes comes from studies with E. coli topoisomerase I and III. Members of type IA group are single subunit Zn++ metalloenzymes recognizing single stranded DNA without high degree of sequence specificity during relaxation reaction of negatively super coiled DNA. So far no inhibitors are known for this group of enzymes inspite of their important role in maintaining homeostasis of DNA topology. Molecular characterization of DNA topoisomerase I from mycobacteria has revealed some of the important features of type IA enzymes hitherto unknown and provide scope for identifying novel inhibitors. The present review describes the recent developments in the area summarizing the distinctive features of mycobacterial topoisomerase I. The enzyme has several properties not shared by either type IA or 113 enzymes with respect to DNA binding, recognition, sequence specificity and interaction pattern. The physiological basis of the unusual features is discussed. The unique properties described would aid in developing the enzyme as a target molecule in pharmaceutical design. In addition, the findings lead to address some fundamental questions on the intracellular role of topoisomerase I in the biology of mycobacteria which are one of the most formidable group of pathogenic organisms.

Helicobacter pylori DprA alleviates restriction barrier for incoming DNA

Helicobacter pylori is a Gram-negative bacterium that colonizes human stomach and causes gastric inflammation. The species is naturally competent and displays remarkable diversity. The presence of a large number of restriction-modification (R-M) systems in this bacterium creates a barrier against natural transformation by foreign DNA. Yet, mechanisms that protect incoming double-stranded DNA (dsDNA) from restriction enzymes are not well understood. A DNA-binding protein, DNA Processing Protein A (DprA) has been shown to facilitate natural transformation of several Gram-positive and Gram-negative bacteria by protecting incoming single-stranded DNA (ssDNA) and promoting RecA loading on it. However, in this study, we report that H. pylori DprA (HpDprA) binds not only ssDNA but also dsDNA thereby conferring protection to both from various exo-nucleases and Type II restriction enzymes. Here, we observed a stimulatory role of HpDprA in DNA methylation through physical interaction with methyltransferases. Thus, HpDprA displayed dual functional interaction with H. pylori R-M systems by not only inhibiting the restriction enzymes but also stimulating methyltransferases. These results indicate that HpDprA could be one of the factors that modulate the R-M barrier during inter-strain natural transformation in H. pylori.

N-Terminal disordered domain of saccharomyces cerevisiae Hop1 protein Is dispensable for DNA binding, bridging, and synapsis of double-stranded DNA molecules but is ecessary for spore formation

The cytological architecture of the synaptonemal complex (SC), a meiosis-specific proteinaceous structure, is evolutionarily conserved among eukaryotes. However, little is known about the biochemical properties of SC components or the mechanisms underlying their roles in meiotic chromosome synapsis and recombination. Functional analysis of Saccharomyces cerevisiae Hop1, a key structural component of SC, has begun to reveal important insights into its function in interhomolog recombination. Previously, we showed that Hop1 is a structure-specific DNA-binding protein, exhibits higher binding affinity for the Holliday junction, and induces structural distortion at the core of the junction. Furthermore, Hop1 promotes DNA condensation and intra- and intermolecular synapsis between duplex DNA molecules. Here, we show that Hop1 possesses a modular domain organization, consisting of an intrinsically disordered N-terminal domain and a protease-resistant C-terminal domain (Hop1CTD). Furthermore, we found that Hop1CTD exhibits strong homotypic as well as heterotypic protein protein interactions, and its biochemical activities were similar to those of the full-length Hop1 protein. However, Hop1CTD failed to complement the meiotic recombination defects of the Delta hop1 strain, indicating that both N- and C-terminal domains of Hop1 are essential for meiosis and spore formation. Altogether, our findings reveal novel insights into the structure-function relationships of Hop1 and help to further our understanding of its role in meiotic chromosome synapsis and recombination.

Detection of Hepatitis B DNA Sequences on Polyelectrolyte Based Non-Covalently Functionalized Flexible Plastic Substrates

Development of simple functionalization methods to attach biomolecules such as proteins and DNA on inexpensive substrates is important for widespread use of low cost, disposable biosensors. Here, we describe a method based on polyelectrolyte multilayers to attach single stranded DNA molecules to conventional glass slides as well as a completely non-standard substrate, namely flexible plastic transparency sheets. We then use the functionalized transparency sheets to specifically detect single stranded Hepatitis B DNA sequences from samples. We also demonstrate a blocking method for reducing non-specific binding of target DNA sequences using negatively charged polyelectrolyte molecules. The polyelectrolyte based functionalization method, which relies on surface charge as opposed to covalent surface linkages, could be an attractive platform to develop assays on inexpensive substrates for low cost biosensing.

Detection of Target DNA using Photo-Reactive Protoporphyrin Moeity on a Nanocomposite Substrate

Detection of pathogens from infected biological samples through conventional process involves cell lysis and purification. The main objective of this work is to minimize the time and sample loss, as well as to increase the efficiency of detection of biomolecules. Electrical lysis of medical sample is performed in a closed microfluidic channel in a single integrated platform where the downstream analysis of the sample is possible. The device functions involve, in a sequence, flow of lysate from lysis chamber passed through a thermal denaturation counter where dsDNA is denatured to ssDNA, which is controlled by heater unit. A functionalized binding chamber of ssDNA is prepared by using ZnO nanorods as the matrix and functionalized with bifunctional carboxylic acid, 16-(2-pyridyldithiol) hexadecanoic acid (PDHA) which is further attached to a linker molecule 1-ethyl-3-(3-dimethylaminopropyl) (EDC). Linker moeity is then covalently bound to photoreactive protoporphyrin (PPP) molecule. The photolabile molecule protoporphyrin interacts with -NH2 labeled single stranded DNA (ssDNA) which thus acts as a probe to detect complimentary ssDNA from target organisms. Thereafter the bound DNA with protoporphyrin is exposed to an LED of particular wavelength for a definite period of time and DNA was eluted and analyzed. UV/Vis spectroscopic analysis at 260/280 nm wavelength confirms the purity and peak at 260 nm is reconfirmed for the elution of target DNA. Quantitative and qualitative data obtained from the current experiments show highly selective detection of biomolecule such as DNA which have large number of future applications in Point-of-Care devices.

DNA Assisted Self-Assembly of PAMAM Dendrimers

We report DNA assisted self-assembly of polyamidoamine (PAMAM) dendrimers using all atom Molecular Dynamics (MD) simulations and present a molecular level picture of a DNA-linked PAMAM dendrimer nanocluster, which was first experimentally reported by Choi et al. (Nano Lett., 2004, 4, 391-397). We have used single stranded DNA (ssDNA) to direct the self-assembly process. To explore the effect of pH on this mechanism, we have used both the protonated (low pH) and nonprotonated (high pH) dendrimers. In all cases studied here, we observe that the DNA strand on one dendrimer unit drives self-assembly as it binds to the complementary DNA strand present on the other dendrimer unit, leading to the formation of a DNA-linked dendrimer dimeric complex. However, this binding process strongly depends on the charge of the dendrimer and length of the ssDNA. We observe that the complex with a nonprotonated dendrimer can maintain a DNA length dependent inter-dendrimer distance. In contrast, for complexes with a protonated dendrimer, the inter-dendrimer distance is independent of the DNA length. We attribute this observation to the electrostatic complexation of a negatively charged DNA strand with the positively charged protonated dendrimer.

Structure-specific nuclease activity of RAGs is modulated by sequence, length and phase position of flanking double-stranded DNA

RAGs (recombination activating genes) are responsible for the generation of antigen receptor diversity through the process of combinatorial joining of different V (variable), D (diversity) and J (joining) gene segments. In addition to its physiological property, wherein RAG functions as a sequence-specific nuclease, it can also act as a structure-specific nuclease leading to genomic instability and cancer. In the present study, we investigate the factors that regulate RAG cleavage on non-B DNA structures. We find that RAG binding and cleavage on heteroduplex DNA is dependent on the length of the double-stranded flanking region. Besides, the immediate flanking double-stranded region regulates RAG activity in a sequence-dependent manner. Interestingly, the cleavage efficiency of RAGs at the heteroduplex region is influenced by the phasing of DNA. Thus, our results suggest that sequence, length and phase positions of the DNA can affect the efficiency of RAG cleavage when it acts as a structure-specific nuclease. These findings provide novel insights on the regulation of the pathological functions of RAGs.

N-Terminal Disordered Domain of Saccharomyces cerevisiae Hop1 Protein Is Dispensable for DNA Binding, Bridging, and Synapsis of Double-Stranded DNA Molecules But Is Necessary for Spore Formation

The cytological architecture of the synaptonemal complex (SC), a meiosis-specific proteinaceous structure, is evolutionarily conserved among eukaryotes. However, little is known about the biochemical properties of SC components or the mechanisms underlying their roles in meiotic chromosome synapsis and recombination. Functional analysis of Saccharomyces cerevisiae Hop1, a key structural component of SC, has begun to reveal important insights into its function in interhomolog recombination. Previously, we showed that Hop1 is a structure-specific DNA-binding protein, exhibits higher binding affinity for the Holliday junction, and induces structural distortion at the core of the junction. Furthermore, Hop1 promotes DNA condensation and intra- and intermolecular synapsis between duplex DNA molecules. Here, we show that Hop1 possesses a modular domain organization, consisting of an intrinsically disordered N-terminal domain and a protease-resistant C-terminal domain (Hop1CTD). Furthermore, we found that Hop1CTD exhibits strong homotypic as well as heterotypic protein protein interactions, and its biochemical activities were similar to those of the full-length Hop1 protein. However, Hop1CTD failed to complement the meiotic recombination defects of the Delta hop1 strain, indicating that both N- and C-terminal domains of Hop1 are essential for meiosis and spore formation. Altogether, our findings reveal novel insights into the structure-function relationships of Hop1 and help to further our understanding of its role in meiotic chromosome synapsis and recombination.

Functional characterization of the precursor and spliced forms of RecA protein of Mycobacterium tuberculosis

The recA locus of pathogenic mycobacteria differs from that of nonpathogenic species because it contains large intervening sequences nested in the RecA homology region that are excised by an unusual protein-splicing reaction. In vivo assays indicated that Mycobacterium tuberculosis recA partially complemented Escherichia coli recA mutants for recombination and mutagenesis. Further, splicing of the 85 kDa precursor to 38 kDa MtRecA protein was necessary for the display of its activity, in vivo. To gain insights into the molecular basis for partial and lack of complementation by MtRecA and 85 kDa proteins, respectively, we purified both of them to homogeneity. MtRecA protein, but not the 85 kDa form, bound stoichiometrically to single-stranded DNA in the presence of ATP. MtRecA protein was cross-linked to 8-azidoadenosine 5'-triphosphate with reduced efficiency, and kinetic analysis of ATPase activity suggested that it is due to decreased affinity for ATP. In contrast, the 85 kDa form was unable to bind ATP, in the presence or absence of ssDNA and, consequently, was entirely devoid of ATPase activity. Molecular modeling studies suggested that the decreased affinity of MtRecA protein for ATP and the reduced efficiency of its hydrolysis might be due to the widening of the cleft which alters the hydrogen bonds and the contact area between the enzyme and the substrate and changes in the disposition of the amino acid residues around the magnesium ion and the gamma-phosphate. The formation of joint molecules promoted by MtRecA protein was stimulated by SSB when the former was added first. The probability of an association between the lack and partial levels of biological activity of RecA protein(s) to that of illegitimate recombination in pathogenic mycobacteria is considered.

Phage lambda beta protein, a component of general recombination, is associated with host ribosomal S1 protein

We have purified phage lambda beta protein produced by a recombinant plasmid carrying bet gene and confirm that it forms a complex with a protein of relative molecular mass 70 kDa. Therefore, beta protein, a component of general genetic recombination, is associated with two functionally diverse complexes; one containing exonuclease and the other 70 kDa protein. Using a number of independent methods, we show that 70 kDa protein is the ribosomal S1 protein of E. coli. Further, the association of 70 kDa protein with beta protein is biologically significant, as the former inhibits joining of the terminal ends of lambda chromosome and renaturation of complementary single stranded DNA promoted by the latter. More importantly, these findings initiate an understanding of an important mode of host- virus interaction in general with specific implication(s) in homologous genetic recombination.

The homologous recombination system of phage lambda. Pairing activities of beta protein

The red genes of phage lambda specify two proteins, exonuclease and beta protein, which are essential for its general genetic recombination in recA- cells. These proteins seem to occur in vivo as an equimolar complex. In addition, beta protein forms a complex with another polypeptide, probably of phage origin, of Mr 70,000. The 70-kDa protein appears to be neither a precursor nor an aggregated form of either exonuclease or beta protein, since antibodies directed against the latter two proteins failed to react with 70-kDa protein on Ouchterlony double diffusion analysis. beta protein promotes Mg2+-dependent renaturation of complementary strands (Kmiec, E., and Holloman, W. K. (1981) J. Biol. Chem. 256, 12636-12639). To look for other pairing activities of beta protein, we developed methods of purification to free it of associated exonuclease. Exonuclease-free beta protein appeared unable to cause the pairing of a single strand with duplex DNA; however, like Escherichia coli single strand binding protein (SSB), beta protein stimulated formation of joint molecules by recA protein from linear duplex DNA and homologous circular single strands. Like recA protein, but unlike SSB, beta protein promoted the joining of the complementary single-stranded ends of phage lambda DNA. beta protein specifically protected single-stranded DNA from digestion by pancreatic DNase. The half-time for renaturation catalyzed by beta protein was independent of DNA concentration, unlike renaturation promoted by SSB and spontaneous renaturation, which are second order reactions. Thus, beta protein resembles recA protein in its ability to bring single-stranded DNA molecules together and resembles SSB in its ability to reduce secondary structure in single-stranded DNA.

Demonstration of IgE Antibodies to Nucleic Acid Antigens in Patients with Sle

Using a combination of avidin-biotin microELISA and solid phase radioimmunoassay, we examined sera from 23 patients with systemic lupus erythematosus (SLE), two patients with established sensitivity to ingested shrimp, and 15 healthy normal subjects. In addition to IgG antibodies, varying amounts of IgE antibodies specific for native DNA (nDNA), denatured or single-stranded DNA (dnDNA), RNA, and tRNA were demonstrable in the sera of SLE patients, but not in the sera of normal subjects. A comparison of the specificity of nucleic acid-specific IgE antibodies present in the sera of shrimp-sensitive patients with those present in the sera of seven SLE patients revealed that the IgE antibodies in the sera of shrimp-sensitive patients specifically recognized shrimp tRNA but not yeast tRNA, calf thymus RNA, or calf thymus DNA, while those present in the sera of patients with SLE recognized all these nucleic acid antigens. The IgE antibodies directed against nDNA, dnDNA, RNA, and tRNA may mediate mast cell and basophil degranulation and thus contribute both to immediate-type hypersensitivity phenomena including hives seen in patients with SLE and to the localization of IgE-nucleic acid complexes in target

Formation of a G-quadruplex at the BCL2 major breakpoint region of the t(14;18) translocation in follicular lymphoma

The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Most breaks on chromosome 18 are located at the 3'-UTR of the BCL2 gene and are mainly clustered in the major breakpoint region (MBR). Recently, we found that the BCL2 MBR has a non-B DNA character in genomic DNA. Here, we show that single-stranded DNA modeled from the template strand of the BCL2 MBR, forms secondary structures that migrate faster on native PAGE in the presence of potassium, due to the formation of intramolecular G-quadruplexes. Circular dichroism shows evidence for a parallel orientation for G-quadruplex structures in the template strand of the BCL2 MBR. Mutagenesis and the DMS modification assay confirm the presence of three guanine tetrads in the structure. 1H nuclear magnetic resonance studies further confirm the formation of an intramolecular G-quadruplex and a representative model has been built based on all of the experimental evidence. We also provide data consistent with the possible formation of a G-quadruplex structure at the BCL2 MBR within mammalian cells. In summary, these important features could contribute to the single-stranded character at the BCL2 MBR, thereby contributing to chromosomal fragility.

Formation of a G-quadruplex at the BCL2 major breakpoint region of the t(14;18) translocation in follicular lymphoma

The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Most breaks on chromosome 18 are located at the 3'-UTR of the BCL2 gene and are mainly clustered in the major breakpoint region (MBR). Recently, we found that the BCL2 MBR has a non-B DNA character in genomic DNA. Here, we show that single-stranded DNA modeled from the template strand of the BCL2 MBR, forms secondary structures that migrate faster on native PAGE in the presence of potassium, due to the formation of intramolecular G-quadruplexes. Circular dichroism shows evidence for a parallel orientation for G-quadruplex structures in the template strand of the BCL2 MBR. Mutagenesis and the DMS modification assay confirm the presence of three guanine tetrads in the structure. 1H nuclear magnetic resonance studies further confirm the formation of an intramolecular G-quadruplex and a representative model has been built based on all of the experimental evidence. We also provide data consistent with the possible formation of a G-quadruplex structure at the BCL2 MBR within mammalian cells. In summary, these important features could contribute to the single-stranded character at the BCL2 MBR, thereby contributing to chromosomal fragility.