P1 Nuclease

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Vepatu Shankar - One of the best experts on this subject based on the ideXlab platform.

  • Single-Strand-Specific Nucleases
    Critical Reviews in Microbiology, 2008
    Co-Authors: S. U. Gite, Vepatu Shankar
    Abstract:

    AbstractSingle-strand-specific Nucleases, which act on single-stranded nucleic acids and single-stranded regions in double-stranded nucleic acids, are multifunctional enzymes and are ubiquitous in distribution. They find wide application as analytical tools in molecular biology research, although enzymes such as P1 Nuclease are also used for production of flavor enhancers such as 5′ IMP and 5′ GMP. Because these enzymes are mainly used as analytical tools, very little attention was paid to aspects relating to their structure-function relationships. However, during the last few years considerable developments have taken place in this area. Single-strand-specific Nucleases, their purification, characteristics, biological role, and applications have been reviewed.

  • Single-strand-specific Nucleases
    Fems Microbiology Reviews, 2003
    Co-Authors: Neelam A. Desai, Vepatu Shankar
    Abstract:

    Single-strand-specific Nucleases are multifunctional enzymes and widespread in distribution. Their ability to act selectively on single-stranded nucleic acids and single-stranded regions in double-stranded nucleic acids has led to their extensive application as probes for the structural determination of nucleic acids. Intracellularly, they have been implicated in recombination, repair and replication, whereas extracellular enzymes have a role in nutrition. Although more than 30 single-strand-specific Nucleases from various sources have been isolated till now, only a few enzymes (S1 Nuclease from Aspergillus oryzae, P1 Nuclease from Penicillium citrinum and Nucleases from Alteromonas espejiana, Neurospora crassa, Ustilago maydis and mung bean) have been characterized to a significant extent. Recently, some of these enzymes have been cloned, their crystal structures solved and their interactions with different substrates have been established. The detection, purification, characteristics, structure–function correlations, biological role and applications of single-strand-specific Nucleases are reviewed.

  • Sing le-St rand-Specif ic N ucleases
    1995
    Co-Authors: S. U. Gite, Vepatu Shankar
    Abstract:

    Single-strand-specific Nucleases, which act on single-stranded nucleic acids and single-stranded regions in double-stranded nucleic acids, are multifunctional enzymes and are ubiquitous in distribution. They find wide application as analytical tools in molecular biology research, although enzymes such as P1 Nuclease are also used for production of flavor enhancers such as 5' IMP and 5' GMP. Because these enzymes are mainly used as analytical tools, very little attention was paid to aspects relating to their structure-function relationships. However, during the last few years considerable developments have taken place in this area. Single-strand-specific Nucleases, their purification, characteristics, biological role, and applications have been reviewed.

  • Single-strand-speci¢c Nucleases
    1995
    Co-Authors: Neelam A. Desai, Vepatu Shankar
    Abstract:

    Single-strand-specific Nucleases are multifunctional enzymes and widespread in distribution. Their ability to act selectively on single-stranded nucleic acids and single-stranded regions in double-stranded nucleic acids has led to their extensive application as probes for the structural determination of nucleic acids. Intracellularly, they have been implicated in recombination, repair and replication, whereas extracellular enzymes have a role in nutrition. Although more than 30 single-strand-specific Nucleases from various sources have been isolated till now, only a few enzymes (S1 Nuclease from Aspergillus oryzae, P1 Nuclease from Penicillium citrinum and Nucleases from Alteromonas espejiana, Neurospora crassa, Ustilago maydis and mung bean) have been characterized to a significant extent. Recently, some of these enzymes have been cloned, their crystal structures solved and their interactions with different substrates have been established. The detection, purification, characteristics, structure^function correlations, biological role and applications of single-strand-specifi

Michael Mitas - One of the best experts on this subject based on the ideXlab platform.

  • Structural properties of Friedreich's ataxia d(GAA) repeats.
    Biochimica et biophysica acta, 1999
    Co-Authors: Iang-shan Suen, Jamie N. Rhodes, Mellisa Christy, Brian R Mcewen, Donald M. Gray, Michael Mitas
    Abstract:

    Abstract The expansion of trinucleotide repeat sequences is the underlying cause of a growing number of inherited human disorders. To provide correlations between DNA structure and mechanisms of trinucleotide repeat expansion, we investigated potential secondary structures formed from the complementary strands of d(GAA·TTC)n, a sequence whose expansion is associated with Friedreich’s ataxia. In 50 mM NaCl, pH 7.5, d(GAA)15 exhibited a cooperative and reversible decrease in large circular dichroism bands at 248 and 272–274 nm over the temperature range of 5–50°C, providing evidence for a base-paired structure at reduced temperatures. Ultraviolet absorbance melting profiles indicated that the melting temperature (Tm) of d(GAA)15 was 40°C. At 5°C, the central portion of d(GAA)15 was hypersensitive to single-strand-specific P1 Nuclease degradation and diethyl pyrocarbonate modification, providing evidence for a hairpin conformation. At temperatures between 25 and 35°C in 50 mM NaCl, the triplet repeat region of d(GAA)15 was uniformly resistant to degradation by P1 Nuclease, including the central portion of the sequence. Our results indicate that the structure of d(GAA)15 is a hairpin at 5°C, unknown but partially base-paired at 37°C, and an approximately random coil above 65°C.

  • At Physiological pH, d(CCG)15 Forms a Hairpin Containing Protonated Cytosines and a Distorted Helix†
    Biochemistry, 1997
    Co-Authors: Maria D. Barron, Ian S. Haworth, Mellisa Christy, Donald M. Gray, Rebecca M. Romero, Barry Gold, Jianli Dai, Michael Mitas
    Abstract:

    To investigate potential structures of d(CGG/CCG)n that might relate to their biological function and association with triplet repeat expansion diseases (TREDs), the structure of a single-stranded (ss) oligonucleotide containing d(CCG)15 [ss(CCG)15] was examined by studies of the pH and temperature dependence of electrophoretic mobility, UV absorbance, circular dichroism, chemical modification, and P1 Nuclease digestion. ss(CCG)15 had an unusually high pKa (7.7 +/- 0.2). At pH 8.5, ss(CCG)15 formed a relatively unstable (Tm = 30 degrees C in 1 mM Na+) hairpin containing CpG base-pair steps. At pH 7.5, the hairpin contained protonated cytosines but no detectable C x +C base pairs, increased thermal stability (Tm = 37 degrees C), increased stacking of the CpG base-pair steps, and a single cytosine that was flipped away from the central portion of the helix. Examination of ss(CCG)18 and ss(CCG)20, which were designed to adopt hairpins containing alternative GpC base-pair steps, revealed hairpins containing CpG base-pair steps, pKas of approximately 8.2 and approximately 8.4, respectively, and distorted helices. The results suggest that DNA sequences containing (CCG)(n > or = 15) adopt hairpin conformations that contain CpG rather than GpC base-pair steps; the mismatched cytosines are protonated at physiological pH but are not H-bonded. We propose that protonation arises from the stacking of two cytosines in the minor groove of a distorted helix.

  • The purine-rich trinucleotide repeat sequences d(CAG) 15 and d(GAC) 15 form hairpins
    Nucleic acids research, 1995
    Co-Authors: Jeffrey Dill, Michael Mitas
    Abstract:

    The structures of single-stranded (ss) oligonucleotides containing (CAG)15 [ss(CAG)15] or (GAC)15 [ss(GAC)15] were examined. At 10 degrees C, the electrophoretic mobilites of the two DNAs were similar to ss(CTG)15, a DNA that forms a hairpin containing base paired and/or stacked thymines. At 37 degrees C in 50 mM NaCl, single-strand-specific P1 Nuclease cleaved the G33-G36 phosphodiesters of ss(GAC)15, and the G32-A34, G35-C36 phosphodiesters of ss(CAG)15 (where the loop apex of both DNAs = A34). Electrophoretic mobility melting profiles indicated that the melting temperature (Tm) of ss(CAG)15 in low (approximately 1 mM Na+) ionic strength was 38 degrees C. In contrast, the Tm of ss(GAC)15 was 49 degrees C, a value similar to the Tm of ss(CTG)15. These results provide evidence that ss(GAC)15 and ss(CAG)15 form similar, but distinguishable hairpin structures.

  • The trinucleotide repeat sequence d(CGG)15 forms a heat-stable hairpin containing Gsyn. Ganti base pairs.
    Biochemistry, 1995
    Co-Authors: Michael Mitas, Jeffrey Dill, Ian S. Haworth
    Abstract:

    To investigate potential structures of d(CGG/CCG)n that might relate to their biological function and association with triplet repeat expansion diseases (TREDs), electrophoretic mobility, chemical modification, and P1 Nuclease studies were performed with a single-stranded (ss) oligonucleotide containing (CGG)15 [ss(CGG)15]. The results suggest that ss(CGG)15 forms a hairpin with the following features: (i) a stem containing Gsyn. Ganti base pairs; (ii) at > or = 200 mM K+, CGG repeats on the 5' portion of the stem base-paired to GCG repeats on the 3' side (referred to as the (b) alignment); and (iii) heat stability (Tm = 75 degrees C in low ionic strength). At < or = 100 mM K+, dimethyl sulfate reactions indicated that the hairpin in the (b) alignment was in equilibrium with another structure, presumably a hairpin in the alternative (a) alignment (CGG repeats on the 5' portion of the stem base-paired to CGG repeats on the 3' portion of the stem). Molecular dynamics simulations suggested that the loop region of the (a) alignment contained two guanines stacked on top of one another. The same guanines in the (b) alignment were base-paired in a syn-anti arrangement. We propose that the stability of the loop partially determines the stem alignment.

  • The trinucleotide repeat sequence d(GTC)15 adopts ahairpin conformation
    Nucleic acids research, 1995
    Co-Authors: Jeffrey Dill, Sara S. Wirth, George Huang, Vincent H.l. Lee, Ian S. Haworth, Michael Mitas
    Abstract:

    The structure of a single-stranded (ss) oligonucleotide containing (GTC)15 [ss(GTC)15] was examined. As a control, parallel studies were performed with ss(CTG)15, an oligonucleotide that forms a hairpin. Electrophoretic mobility, KMnO4 oxidation and P1 Nuclease studies demonstrate that, similar to ss(CTG)15, ss(GTC)15 forms a hairpin containing base paired and/or stacked thymines in the stem. Electrophoretic mobility melting profiles performed in approximately 1 mM Na+ revealed that the melting temperature of ss(GTC)15 and ss(CTG)15 were 38 and 48 degrees C respectively. The loop regions of ss(GTC)15 and ss(CTG)15 were cleaved by single-strand-specific P1 Nuclease at the T25-C29 and G26-C27 phosphodiester bonds respectively (where the loop apex of the DNAs is T28). Molecular dynamics simulations suggested that in ss(GTC)15 the loop was bent towards the major groove of the stem, apparently causing an increased exposure of the T25-C29 region to solvent. In ss(CTG)15 guanine--guanine stacking caused a separation of the G26 and C27 bases, resulting in exposure of the intervening phosphodiester to solvent. The results suggest that ss(GTC)15 and ss(CTG)15 form similar, but distinguishable, hairpin structures.

Ian S. Haworth - One of the best experts on this subject based on the ideXlab platform.

  • Stability and strand asymmetry in the non-B DNA structure at the bcl-2 major breakpoint region.
    The Journal of biological chemistry, 2004
    Co-Authors: Sathees C. Raghavan, Ian S. Haworth, Sabrina I. Houston, Balachandra G. Hegde, Ralf Langen, Michael R. Lieber
    Abstract:

    The t(14;18) translocation involving the Ig heavy chain locus and the BCL-2 gene is the single most common chromosomal translocation in human cancer. Recently we reported in vitro and in vivo chemical probing data indicating that the 150-bp major breakpoint region (Mbr), which contains three breakage subregions (hotspots) (known as peaks I, II, and III), has single-stranded character and hence a non-B DNA conformation. Although we could document the non-B DNA structure formation at the bcl-2 Mbr, the structural studies were limited to chemical probing. Therefore, in the present study, we used multiple methods including circular dichroism to detect the non-B DNA at the bcl-2 Mbr. We established a new gel shift method to detect the altered structure at neutral pH on shorter DNA fragments containing the bcl-2 Mbr and analyzed the fine structural features. We found that the single-stranded region in the non-B DNA structure observed is stable for days and is asymmetric with respect to the Watson and Crick strands. It could be detected by oligomer probing, a bisulfite modification assay, or a P1 Nuclease assay. We provide evidence that two different non-B conformations exist at peak I in addition to the single one observed at peak III. Finally we used mutagenesis and base analogue incorporation to show that the non-B DNA structure formation requires Hoogsteen pairing. These findings place major constraints on the location and nature of the non-B conformations assumed at peaks I and III of the bcl-2 Mbr.

  • At Physiological pH, d(CCG)15 Forms a Hairpin Containing Protonated Cytosines and a Distorted Helix†
    Biochemistry, 1997
    Co-Authors: Maria D. Barron, Ian S. Haworth, Mellisa Christy, Donald M. Gray, Rebecca M. Romero, Barry Gold, Jianli Dai, Michael Mitas
    Abstract:

    To investigate potential structures of d(CGG/CCG)n that might relate to their biological function and association with triplet repeat expansion diseases (TREDs), the structure of a single-stranded (ss) oligonucleotide containing d(CCG)15 [ss(CCG)15] was examined by studies of the pH and temperature dependence of electrophoretic mobility, UV absorbance, circular dichroism, chemical modification, and P1 Nuclease digestion. ss(CCG)15 had an unusually high pKa (7.7 +/- 0.2). At pH 8.5, ss(CCG)15 formed a relatively unstable (Tm = 30 degrees C in 1 mM Na+) hairpin containing CpG base-pair steps. At pH 7.5, the hairpin contained protonated cytosines but no detectable C x +C base pairs, increased thermal stability (Tm = 37 degrees C), increased stacking of the CpG base-pair steps, and a single cytosine that was flipped away from the central portion of the helix. Examination of ss(CCG)18 and ss(CCG)20, which were designed to adopt hairpins containing alternative GpC base-pair steps, revealed hairpins containing CpG base-pair steps, pKas of approximately 8.2 and approximately 8.4, respectively, and distorted helices. The results suggest that DNA sequences containing (CCG)(n > or = 15) adopt hairpin conformations that contain CpG rather than GpC base-pair steps; the mismatched cytosines are protonated at physiological pH but are not H-bonded. We propose that protonation arises from the stacking of two cytosines in the minor groove of a distorted helix.

  • The trinucleotide repeat sequence d(CGG)15 forms a heat-stable hairpin containing Gsyn. Ganti base pairs.
    Biochemistry, 1995
    Co-Authors: Michael Mitas, Jeffrey Dill, Ian S. Haworth
    Abstract:

    To investigate potential structures of d(CGG/CCG)n that might relate to their biological function and association with triplet repeat expansion diseases (TREDs), electrophoretic mobility, chemical modification, and P1 Nuclease studies were performed with a single-stranded (ss) oligonucleotide containing (CGG)15 [ss(CGG)15]. The results suggest that ss(CGG)15 forms a hairpin with the following features: (i) a stem containing Gsyn. Ganti base pairs; (ii) at > or = 200 mM K+, CGG repeats on the 5' portion of the stem base-paired to GCG repeats on the 3' side (referred to as the (b) alignment); and (iii) heat stability (Tm = 75 degrees C in low ionic strength). At < or = 100 mM K+, dimethyl sulfate reactions indicated that the hairpin in the (b) alignment was in equilibrium with another structure, presumably a hairpin in the alternative (a) alignment (CGG repeats on the 5' portion of the stem base-paired to CGG repeats on the 3' portion of the stem). Molecular dynamics simulations suggested that the loop region of the (a) alignment contained two guanines stacked on top of one another. The same guanines in the (b) alignment were base-paired in a syn-anti arrangement. We propose that the stability of the loop partially determines the stem alignment.

  • The trinucleotide repeat sequence d(GTC)15 adopts ahairpin conformation
    Nucleic acids research, 1995
    Co-Authors: Jeffrey Dill, Sara S. Wirth, George Huang, Vincent H.l. Lee, Ian S. Haworth, Michael Mitas
    Abstract:

    The structure of a single-stranded (ss) oligonucleotide containing (GTC)15 [ss(GTC)15] was examined. As a control, parallel studies were performed with ss(CTG)15, an oligonucleotide that forms a hairpin. Electrophoretic mobility, KMnO4 oxidation and P1 Nuclease studies demonstrate that, similar to ss(CTG)15, ss(GTC)15 forms a hairpin containing base paired and/or stacked thymines in the stem. Electrophoretic mobility melting profiles performed in approximately 1 mM Na+ revealed that the melting temperature of ss(GTC)15 and ss(CTG)15 were 38 and 48 degrees C respectively. The loop regions of ss(GTC)15 and ss(CTG)15 were cleaved by single-strand-specific P1 Nuclease at the T25-C29 and G26-C27 phosphodiester bonds respectively (where the loop apex of the DNAs is T28). Molecular dynamics simulations suggested that in ss(GTC)15 the loop was bent towards the major groove of the stem, apparently causing an increased exposure of the T25-C29 region to solvent. In ss(CTG)15 guanine--guanine stacking caused a separation of the G26 and C27 bases, resulting in exposure of the intervening phosphodiester to solvent. The results suggest that ss(GTC)15 and ss(CTG)15 form similar, but distinguishable, hairpin structures.

  • Hairpin properties of single-stranded DNA containing a GC-rich triplet repeat: (CTG) 15
    Nucleic acids research, 1995
    Co-Authors: Michael Mitas, Jeffrey Dill, Timothy J. Kamp, Eric J. Chambers, Ian S. Haworth
    Abstract:

    Although triplet repeat DNA sequences are scattered throughout the human genome, their biological function remains obscure. To aid in correlating potential structures of these nucleic acids with their function, we propose their classification based on the presence or absence of a palindromic dinucleotide within the triplet, the G + C content, and the presence or absence of a homopolymer. Five classes of double-stranded (ds) triplet repeats are distinguished. Class I repeats, which are defined by the presence of a GC or CG palindrome, have the lowest base stacking energies, exhibit the lowest rates of slippage synthesis [Schlotterer and Tautz (1992) Nucleic Acids Res., 20, 211] and are uniquely associated with triplet repeat expansion diseases. The six single-stranded (ss) triplet repeats within Class I also have the potential to form hairpin structures, as determined by energy minimization. To explore the possibility of hairpin formation by ss Class I triplet repeats, studies were performed with a ss oligonucleotide containing 15 prototypic CTG repeats [ss (CTG)15]. Electrophoretic, P1 Nuclease and KMnO4 oxidation data demonstrate that ss (CTG)15 forms a hairpin containing base paired and/or stacked thymines in the stem. Potential functions of hairpins containing Class I triplet repeats are discussed with respect to protein translation and mRNA splicing. Further, potential roles of hairpin structures in triplet repeat expansion events are discussed.

Charles M. Radding - One of the best experts on this subject based on the ideXlab platform.

  • Interactions of three strands in joints made by RecA protein.
    Biochemistry, 1993
    Co-Authors: Sung-kay Chiu, Basuthkar J. Rao, R. M. Story, Charles M. Radding
    Abstract:

    RecA protein from Escherichia coli has been used to form a triple-stranded DNA structure from either single-stranded M13 DNA or a single-stranded oligonucleotide plus a duplex oligonucleotide with a hairpin loop. The secondary structure of purified deproteinized triplex was examined by probing with DNase I, P1 Nuclease, potassium permanganate, and diethyl pyrocarbonate. The two strands destined to form heteroduplex DNA showed the same patterns of chemical modification and enzymatic digestion as control duplex DNA, indicating that they formed a normal duplex substructure. However, the nascent outgoing strand showed properties consistent with a novel triplex structure: most of its purine residues, especially adenines, were hyperreactive to all probes. The patterns of digestion by DNase I and P1 Nuclease indicated that the nascent outgoing strand was not a freely mobile or single-stranded branch but rather was still interacting with the newly formed heteroduplex DNA. On the basis of the planar base triads proposed previously (Rao et al., 1993) and energy minimization of a third strand in the major groove of B-form DNA, we derived a model that helps to rationalize the properties revealed by chemical and enzymatic probing.

  • Torsional stress generated by RecA protein during DNA strand exchange separates strands of a heterologous insert.
    Proceedings of the National Academy of Sciences of the United States of America, 1992
    Co-Authors: Biru Jwang, Charles M. Radding
    Abstract:

    Previous studies have shown that the helical RecA nucleoprotein filament formed on a circular single strand of DNA causes the progressive, directional transfer of a complementary strand from naked linear duplex DNA to the nucleoprotein filament, even when the duplex contains a sizable heterologous insertion. Since RecA protein lacks demonstrable helicase activity, the mechanism by which it pushes strand exchange through long heterologous inserts has been a quandary. In the present study, a linear duplex substrate with an insertion of 110 base pairs in its middle yielded the expected products, whereas much less of the heteroduplex product was seen when the insertion was located at either end of the duplex substrate or 160 base pairs from the far end of the duplex substrate. In an ongoing reaction of the substrate with an insertion in its middle, P1 Nuclease cleaved intermediates from the point of the insertion to various distal sites. Acting on a duplex substrate that contained a single nick located in the complementary strand just beyond the insertion, RecA protein formed joint molecules but failed to complete strand exchange. These data show that negative torsional stress is generated by distant homologous interactions that occur beyond the heterologous insertion and that such stress is essential for unwinding a heterologous insertion that otherwise halts strand exchange.

  • Stable three-stranded DNA made by RecA protein.
    Proceedings of the National Academy of Sciences of the United States of America, 1991
    Co-Authors: Basuthkar J. Rao, M Dutreix, Charles M. Radding
    Abstract:

    Abstract When RecA protein, in the form of a nucleoprotein filament containing circular single-stranded DNA (plus strand only), reacts with homologous linear duplex DNA, a directional transfer ensues of a strand from the duplex DNA to the nucleoprotein filament, resulting in the displacement of the linear plus strand in the 5' to 3' direction. The initial homologous synapsis, however, can occur at either end of the duplex DNA, or anywhere in between, and when homology is restricted to different regions of the duplex DNA, the joint molecules that form in each region show striking differences in stability upon deproteinization: distal joints greater than proximal joints much greater than medial joints. In the deproteinized distal joints, which are thermostable, 2000 nucleotide residues of the circular plus strand are resistant to P1 Nuclease; both strands of the original duplex DNA remain resistant to P1 Nuclease, and the potentially displaceable linear plus strand, which has a 3' homologous end, remains resistant to Escherichia coli exoNuclease I. These observations suggest that RecA protein promotes homologous pairing and strand exchange via long three-stranded DNA intermediates and, moreover, that, once formed, such triplex structures in natural DNA are stable even when RecA protein has been removed.

David Roth - One of the best experts on this subject based on the ideXlab platform.

  • Hairpin opening by single-strand-specific Nucleases
    Nucleic Acids Research, 1995
    Co-Authors: Elena B. Kabotyanski, Chengming Zhu, Deborah A. Kallick, David Roth
    Abstract:

    DNA molecules with covalently sealed (hairpin) ends are probable intermediates in V(D)J recombination. According to current models hairpin ends are opened to produce short single-stranded extensions that are thought to be precursors of a particular type of extra nucleotides, termed P nucleotides, which are frequently present at recombination junctions. Nothing is known about the activities responsible for hairpin opening. We have used two single-strand-specific Nucleases to explore the effects of loop sequence on the hairpin opening reaction. Here we show that a variety of hairpin ends are opened by P1 Nuclease and mung bean Nuclease (MBN) to leave short, 1-2 nt single-stranded extensions. Analysis of 22 different hairpin sequences demonstrates that the terminal 4 nt of the hairpin loop strongly influence the sites of cleavage. Correlation of the Nuclease digestion patterns with structural (NMR) data for some of the hairpin loops studied here provides new insights into the structural features recognized by these enzymes.