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B J Rao

Publications and source records attributed to B J Rao.

34 records · Page 2Linked to original sources
PubMed1993

Interactions of three strands in joints made by RecA protein.

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.

Bacteriophage M13↗
PubMed1993

Homologous recognition promoted by RecA protein via non-Watson-Crick bonds between identical DNA strands.

The RecA protein of Escherichia coli forms a nucleoprotein filament that promotes homologous recognition and subsequent strand exchange between a single strand and duplex DNA via a three-stranded intermediate. Recognition of homology within three-stranded nucleoprotein complexes, which is probably central to genetic recombination, is not well understood as compared with the mutual recognition of complementary single strands by Watson-Crick base pairing. Using oligonucleotides, we examined the determinants of homologous recognition within RecA nucleoprotein filaments. Filaments that contained a single strand of DNA recognized homology not only in a complementary oligonucleotide but also in an identical oligonucleotide, whether their respective sugar-phosphate backbones were antiparallel or parallel, and a filament that contained duplex DNA showed the same polymorphic versatility in the recognition of homology. Recognition of self by a filament that contains a single strand reveals that RecA filaments can recognize homology via non-Watson-Crick hydrogen bonds. Recognition of multiple forms of the same sequence by duplex DNA in the filament shows that it primarily senses base-sequence homology, and suggests that recognition can be accomplished prior to the establishment of new Watson-Crick base pairs in heteroduplex products. However, unlike the initial recognition of homology, strand exchange is stereospecific, requiring the proper antiparallel orientation of complementary strands.

Bacteriophage M13↗
PubMed1993

Homologous recognition and triplex formation promoted by RecA protein between duplex oligonucleotides and single-stranded DNA.

RecA protein formed a stable triplex from a 33 bp duplex oligonucleotide and a circular plus strand of M13 DNA when a hairpin connection at the proximal end of the homologous duplex oligonucleotide blocked displacement of the 5' end of its own plus strand. An oligonucleotide with a hairpin connection at the other end yielded five times fewer joints that survived deproteinization, and an ordinary duplex oligonucleotide yielded none. The stability of the three-stranded structure was not attributable to exonucleolytic nibbling of the 3' end of the hairpin oligonucleotide, which could generate a region of stable duplex DNA. In the triplexes, the hairpin duplex became more accessible to copper phenanthroline, exhibited novel sites of cleavage by DNase I, and resisted digestion by Escherichia coli exonuclease I. The enzymatic methylation of only two residues at N-6 adenine and two at N-4 cytosine in the hairpin duplex prior to the pairing reaction lowered the tm of triplexes by 8 deg.C, whereas extensive methylation at N-7 guanine by dimethyl sulfate had no effect. These results are discussed in relation to possible models of triplex DNA.

Adenine↗
PubMed1992

RNA-DNA hybridization promoted by E. coli RecA protein.

RecA protein of E. coli plays a central regulatory role that is induced by damage to DNA and results in the inactivation of LexA repressor. In vitro, RecA protein binds preferentially to single-stranded DNA to form a nucleoprotein filament that can recognize homology in naked duplex DNA and promote extensive strand exchange. Although RecA protein shows little tendency at neutral pH to bind to RNA, we found that it nonetheless catalyzed at 37 degrees C the hybridization of complementary RNA and single-stranded DNA sequences. Hybrids made by RecA protein at 37 degrees C appeared indistinguishable from ones prepared by thermal annealing. RNA-DNA hybridization by RecA protein at neutral pH required, as does RecA-promoted homologous pairing, optimal conditions for the formation of RecA nucleoprotein filaments. The cosedimentation of RNA with those filaments further paralleled observations made on the formation of networks of nucleoprotein filaments with double-stranded DNA, an instrumental intermediate in homologous pairing in vitro. These similarities with the pairing reaction support the view that RecA protein acts specifically in the hybridization reaction.

Bacterial Proteins↗
PubMed1991

The effects on strand exchange of 5' versus 3' ends of single-stranded DNA in RecA nucleoprotein filaments.

Since the ends of DNA chains are thought to be important in homologous recombination, the way in which RecA protein and similar recombination enzymes process ends is important. We analyzed the effects of ends both on the formation of joints, and the progression of strand exchange. When the only homologous end was provided by a single strand, there was no significant difference between the formation of joints at a 5' end or a 3' end; but in agreement with the report of Konforti & Davis, Escherichia coli single-stranded DNA binding protein (SSB) selectively inhibited the activity of 5' ends. Complete strand exchange, assessed by study of linear single-stranded and double-stranded substrates, took place only in the 5' to 3' direction relative to DNA in the nucleoprotein filament. These observations pose a paradox: in the presence of SSB, of which there are about 800 tetramers per cell, the formation of homologous joints by RecA protein is favored at a 3' end, from which, however, authentic strand exchange appears not to occur. Since observations reported here and elsewhere show that joints have different properties when formed at a 5' versus a 3' end, we suggest that they may be processed differently in vivo.

DNA, Circular↗
PubMed1991

Stable three-stranded DNA made by RecA protein.

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.

DNA↗
PubMed1991

Production of triple-stranded recombination intermediates by RecA protein, in vitro.

During the directional strand exchange that is promoted by RecA protein between linear duplex DNA and circular single-stranded DNA, a triple-stranded DNA intermediate was formed and persisted even after the completion of strand transfer followed by deproteinization. In the deproteinized three-stranded DNA complexes, the sequestered linear third strand resisted digestion by E coli exonuclease I. In relation to polarity of strand exchange which defines the proximal and distal ends of the duplex DNA, when homology was restricted to the distal region of duplex substrate, the joints formed efficiently and were stable even upon complete deproteinization. Enzymatic probing of deproteinized distal joints with nuclease P1 revealed that the joints consist of long three-stranded structures that at neutral pH lack significant single-stranded character in any of the three strands. Instead of circular single-stranded DNA, when a linear single strand is recombined with partially homologous duplex DNA, in the presence of SSB, the formation of homologous joints by RecA protein, is significantly more efficient at distal end than at the proximal. Taken together, these observations suggest that with any single-stranded DNA (circular or linear), RecA protein efficiently promotes the formation of distal joints, from which, however, authentic strand exchange may not occur. Moreover, these joints might represent an intermediate which is trapped into a stable triple stranded state.

DNA Replication↗
PubMed1990

RecA protein reinitiates strand exchange on isolated protein-free DNA intermediates. An ADP-resistant process.

Efficient homologous pairing de novo of linear duplex DNA with a circular single strand (plus strand) coated with RecA protein requires saturation and extension of the single strand by the protein. However, strand exchange, the transfer of a strand from duplex DNA to the nucleoprotein filament, which follows homologous pairing, does not require the stable binding of RecA protein to single-stranded DNA. When RecA protein was added back to isolated protein-free DNA intermediates in the presence of sufficient ADP to inhibit strongly the binding of RecA protein to single-stranded DNA, strand exchange nonetheless resumed at the original rate and went to completion. Characterization of the protein-free DNA intermediate suggested that it has a special site or region to which RecA protein binds. Part of the nascent displaced plus strand of the deproteinized intermediate was unavailable as a cofactor for the ATPase activity of RecA protein, and about 30% resisted digestion by P1 endonuclease, which acts preferentially on single-stranded DNA. At the completion of strand exchange, when the distal 5' end of the linear minus strand had been fully incorporated into heteroduplex DNA, a nucleoprotein complex remained that contained all three strands of DNA from which the nascent displaced strand dissociated only over the next 50 to 60 minutes. Deproteinization of this intermediate yielded a complex that also contained three strands of DNA in which the nascent displaced strand was partially resistant to both Escherichia coli exonuclease I and P1 endonuclease. The deproteinized complex showed a broad melting transition between 37 degrees C and temperatures high enough to melt duplex DNA. These results show that strand exchange can be subdivided into two stages: (1) the exchange of base-pairs, which creates a new heteroduplex pair in place of a parental pair; and (2) strand separation, which is the physical displacement of the unpaired strand from the nucleoprotein filament. Between the creation of new heteroduplex DNA and the eventual separation of a third strand, there exists an unusual DNA intermediate that may contain three-stranded regions of natural DNA that are several thousand bases in length.

Adenosine Diphosphate↗
PubMed1988

Reversibility of strand invasion promoted by recA protein and its inhibition by Escherichia coli single-stranded DNA-binding protein or phage T4 gene 32 protein.

When recA protein promotes homologous pairing and strand exchange involving circular single strands and linear duplex DNA, the protein first polymerizes on the single-stranded DNA to form a nucleoprotein filament which then binds naked duplex DNA to form nucleoprotein networks, the existence of which is independent of homology, but requires the continued presence of recA protein (Tsang, S. S., Chow, S. A., and Radding, C. M. (1985) Biochemistry 24, 3226-3232). Further experiments revealed that within a few minutes after the beginning of homologous pairing and strand exchange, these networks began to be replaced by a distinct set of networks with inverse properties: their formation depended upon homology, but they survived removal of recA protein by a variety of treatments. Formation of this second kind of network required that homology be present specifically at the end of the linear duplex molecule from which strand exchange begins. Escherichia coli single-stranded DNA-binding protein or phage T4 gene 32 protein largely suppressed the formation of this second population of networks by inactivating the newly formed heteroduplex DNA, which, however, could be reactivated when recA protein was dissociated by incubation at 0 degrees C. We interpret these observations as evidence of reinitiation of strand invasion when recA protein acts in the absence of auxiliary helix-destabilizing proteins. These observations indicate that the nature of the nucleoprotein products of strand exchange determines whether pairing and strand exchange are reversible or not, and they further suggest a new explanation for the way in which E. coli single-stranded DNA-binding protein and gene 32 protein accelerate the apparent forward rate of strand exchange promoted by recA protein, namely by suppressing initiation of the reverse reaction.

Cold Temperature↗
PubMed1987

DNase I site mapping and micrococcal nuclease digestion of pachytene chromatin reveal novel structural features.

A comparison of the DNase I digestion products of the 32P-5'-end-labeled pachytene nucleosome core particles (containing histones H2A, TH2A, X2, H2B, TH2B, H3, and H4) and liver nucleosome core particles (containing somatic histones H2A, H2B, H3, and H4) revealed that the cleavage sites that are 30, 40, and 110 nucleotides away from the 5'-end are significantly more accessible in the pachytene core particles than in the liver core particles. These cleavage sites correspond to the region wherein H2B interacts with the nucleosome core DNA. These results, therefore, suggest that the histone-DNA interaction at these sites in the pachytene core particles is weaker, possibly because of the presence of the histone variant TH2B interacting at similar topological positions in the nucleosome core as that of its somatic counterpart H2B. Such a loosened structure may also be maintained even in the native pachytene chromatin since micrococcal nuclease digestion of pachytene nuclei resulted in a higher ratio of subnucleosomes (SN4 + SN7) to mononucleosomes than that observed in liver chromatin.

Animals↗
PubMed1986

Ability of RecA protein to promote a search for rare sequences in duplex DNA.

RecA nucleoprotein filaments found homologous targets even when the latter was mixed with 200,000 times as much heterologous duplex DNA. By contrast, mixing of the single-stranded probe with only 100 times as much heterologous single strands markedly reduced the rate of finding homologous duplex molecules. Titration of the reaction with different proportions of homologous single-stranded DNA distinguished a condition under which the search for homology itself was rate limiting from a condition under which some later step was limiting. Less than 1 min was required to scan 6.4 kilobase pairs of duplex DNA for homology to a RecA-coated single strand of the same size, but these experiments revealed that rapid searching by RecA nucleoprotein filaments was largely confined to neighboring duplex molecules. These observations provide guidelines for the use of RecA protein in locating rare sequences in complex mixtures of duplex DNA, and we describe a simple protocol by which rare sequences can be rapidly enriched at least a thousandfold.

Adenosine Triphosphate↗
PubMed1983

Structural organization of the meiotic prophase chromatin in the rat testis.

Pachytene nuclei were isolated from rat testes by the unit gravity sedimentation technique and contained histone variants H1a, H1t, TH2A, TH2B, and X2 in addition to the somatic histones H1bde, H1c, H2A, H2B, H3, and H4. The basic organization of the pachytene chromatin namely the nucleosome repeat length and the accessibility to micrococcal nuclease, was similar to that of rat liver interphase chromatin. However, when digested by DNase I, the susceptibility of pachytene chromatin was 25% more than liver chromatin under identical conditions. Nucleosome core particles were isolated from both liver and pachytene nuclei and were characterized for their DNA length and integrity of the nucleoprotein on low ionic strength nucleoprotein gels. While liver core particles contained all the somatic histones H2A, H2B, H3, and H4, in the pachytene core particles, histone variants TH2A, X2, and TH2B had replaced nearly 60% of the respective somatic histones. A comparison of the circular dichroism spectra obtained for pachytene and liver core particles indicated that the pachytene core particles were less compact than the liver core particles. Studies on the thermal denaturation properties of the two types of core particles revealed that the fraction of the pachytene core DNA melting at the premelting temperature region of 55-60 degrees C was significantly higher than that of the liver core DNA.

Animals↗
PubMed1982

Localization of testis-variant histones in rat testis chromatin.

Nucleosome core particles and oligonucleosomes were isolated by digesting rat testis nuclei with micrococcal nuclease to 20% acid-solubility, followed by fractionation of the digest on a Bio-Gel A-5m column. The core particles thus isolated were characterized on the basis of their DNA length of 151 +/- 5 base-pairs and sedimentation coefficient of 11.4S. Analysis of the acid-soluble proteins of the core particles indicated that histones TH2B and X2 are constituents of the core particles, in addition to the somatic histones H2A, H2B, H3 and H4. The acid-soluble proteins of the oligonucleosomes comprised all the histones, including both the somatic (H1, H2A, H2B, H3, H4 and X2) and the testis-specific ones (TH1 and TH2B). It was also observed that histones TH1 and H1 are absent from the core particles and were readily extracted from the chromatin by 0.6 M-NaCl, which indicated that both of them are bound to the linker DNA.

Animals↗
PubMed1980

Ungual tufts in the follow-up of patients on maintenance hemodialysis.

A recent publication showed the earliest signs of renal osteodystrophy in patients on maintenance hemodialysis (MHD) to occur in the tufts [1]. We present three patients on MHD, in two of whom the earliest unequivocal radiographic improvement was seen in the tufts. The third patient had been on MHD and, following subtotal parathyroidectomy, showed significant osseous improvement in the hands with relatively equal improvement in the hands with relatively equal improvement in the tufts and middle phalanges. It appears that, with the use of routine primary or secondary magnification, the tufts are radiographically a highly sensitive site for both the early occurrence and improvement of osteodystrophy in patients on MHD. We are not aware of any previous publication pointing out exclusive improvement in the tufts in patients on MHD with virtually no change in the mid-phalanges. Two of these three cases illustrate that renal osteodystrophy is reversible by maintenance hemodialysis alone.

Chronic Kidney Disease-Mineral and Bone Disorder↗
PubMedDate not supplied

Richter's hernia: a rare presentation of abdominal tuberculosis.

Intestinal obstruction due to Richter's hernia has not been reported in abdominal tuberculosis. This 21-year-old man with abdominal tuberculosis presented with small gut obstruction due to Richter's hernia associated with ascites and patent right processus vaginalis (PV). He underwent laparotomy with reduction of Richter's hernia and closure of the PV at the right deep inguinal ring.

Adult↗