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B J Rao

Publications and source records attributed to B J Rao.

At least 19 recordsLinked to original sources
PubMed2005

DNA binding and pairing activity of OsDmc1, a recombinase from rice.

A cloned cDNA corresponding to OsDMC1 from rice anther tissue was expressed in Escherichia coli. The OsDmc1 protein was largely present in the inclusion bodies of the cell lysatE., which was solubilized by 8.0 M urea containing buffeR., purified to homogeneity by Ni-CAM agarose column chromatography, followed by renaturation to its native state through stepwise dialysis against reduced concentrations of urea. The purified protein cross-reacted with anti-yeast Dmc1 antibodies. The binding efficiency observed with circular single-stranded DNA (ssDNA) was similar to that with circular double-stranded DNA (dsDNA). The binding to either DNA showed no ATP dependencE., but required 5-10 mM Mg2+ in the presence of ATP. Even though the protein binding to dsDNA was as efficient as it was to ssDNA, the former induced no DNA dependent ATPasE., whereas the binding to ssDNA stimulated a significant level of DNA dependent ATPase activity. OsDmc1-ssDNA complex, with its ATPase proficiency, also mediated renaturation of homologous complementary strands as well as assimilation of single strands into homologous supercoiled duplexes leading to D-loop formation. The D-loop formation was lowered by excess of OsDmc1 protein. This D-loop formation activity was promoted by non-hydrolyzable ATP analog, AMP-PNP and was not observed in absence of ATP or presence of ADP/ATP-gamma-S. These properties reflected the classical hallmarks of a recombinase and represented the first biochemical characterization of a plant Dmc1 protein.

Adenosine Triphosphatases↗
PubMed2001

Incoming nucleotide binds to Klenow ternary complex leading to stable physical sequestration of preceding dNTP on DNA.

Klenow-DNA complex is known to undergo a rate-limiting, protein conformational transition from an 'open' to 'closed' state, upon binding of the 'correct' dNTP at the active site. In the 'closed' state, Mg(2+) mediates a rapid chemical step involving nucleophilic displacement of pyrophosphate by the 3' hydroxyl of the primer terminus. The enzyme returns to the 'open' state upon the release of PPi and translocation permits the next round of reaction. To determine whether Klenow can translocate to the next site on the addition of the next dNTP, without the preceding chemical step, we studied the ternary complex (Klenow-DNA-dNTP) in the absence of Mg(2+). While the ternary complex is proficient in chemical addition of dNTPs in Mg(2+), as revealed by primer extensions, the same in Mg(2+)-deficient conditions lead to non-covalent (physical) sequestration of first two 'correct' dNTPs in the ternary complex. Moreover, the second dNTP traps the first one in the DNA-helix of the ternary complex. Such a dNTP-DNA complex is found to be stable even after the dissociation of KLENOW: This reveals the novel state of the dNTP-DNA complex where the complementary base is stacked in a DNA-helix non-covalently, without the phosphodiester linkage. Further, shuttling of the DNA between the polymerase and the exonuclease site mediates the release of such a DNA complex. Interestingly, Klenow in such a Mg(2+)-deficient ternary complex exhibits a 'closed' conformation.

Binding Sites↗
PubMed2001

Real time fluorescence analysis of the RecA filament: implications of base pair fluidity in repeat realignment.

During recombination, when Escherichia coli RecA mediates annealing across DNA repeats, Watson-Crick chemistry can only specify the complementarity of pairing, but not the most optimal frame of alignment. We describe that although stochastic alignments across poly(dA) and poly(dT) can lead to sub-optimally annealed duplexes containing ssDNA gaps/overhangs, the same are realigned into an optimal frame by a putative motor activity of RecA [Sen et al. (2000) Biochemistry 39, 10196-10206]. In the present study, we analyze the nature of realignment intermediates in real time, by employing a fluorescent probe, 2-aminopurine (2AP), which can not only report the status of RecA on the unstacked duplex, but also the fluidity of bases in such a filament. Although known to display a lower affinity for duplex DNA, RecA seems to remain functionally associated with these sub-optimally aligned repeat duplexes, until the realignment approaches completion. Moreover, a comparison of 2AP fluorescence in repeat versus mixed sequences indicates that bases in a RecA repetitive DNA filament exhibit higher degrees of freedom that might mediate a 'non-planar hydrogen bonding cross talk' across the bases on either strand. We discuss a model to explain the mechanistic basis of realignment and its implications in signaling the end of homology maximization, which triggers RecA fall off.

2-Aminopurine↗
PubMed2001

Local repeat sequence organization of an intergenic spacer in the chloroplast genome of Chlamydomonas reinhardtii leads to DNA expansion and sequence scrambling: a complex mode of "copy-choice replication"?

Parent-specific, randomly amplified polymorphic DNA (RAPD) markers were obtained from total genomic DNA of Chlamydomonas reinhardtii. Such parent-specific RAPD bands (genomic fingerprints) segregated uniparentally (through mt+) in a cross between a pair of polymorphic interfertile strains of Chlamydomonas (C. reinhardtii and C. minnesotti), suggesting that they originated from the chloroplast genome. Southern analysis mapped the RAPD-markers to the chloroplast genome. One of the RAPD-markers, "P2" (1.6 kb) was cloned, sequenced and was fine mapped to the 3 kb region encompassing 3' end of 23S, full 5S and intergenic region between 5S and psbA. This region seems divergent enough between the two parents, such that a specific PCR designed for a parental specific chloroplast sequence within this region, amplified a marker in that parent only and not in the other, indicating the utility of RAPD-scan for locating the genomic regions of sequence divergence. Remarkably, the RAPD-product, "P2" seems to have originated from a PCR-amplification of a much smaller (about 600 bp), but highly repeat-rich (direct and inverted) domain of the 3 kb region in a manner that yielded no linear sequence alignment with its own template sequence. The amplification yielded the same uniquely "sequence-scrambled" product, whether the template used for PCR was total cellular DNA, chloroplast DNA or a plasmid clone DNA corresponding to that region. The PCR product, a "unique" new sequence, had lost the repetitive organization of the template genome where it had originated from and perhaps represented a "complex path" of copy-choice replication.

Animals↗
PubMed2001

MutS recognition: multiple mismatches and sequence context effects.

Escherichia coli MutS is a versatile repair protein that specifically recognizes not only various types of mismatches but also single stranded loops of up to 4 nucleotides in length. Specific binding, followed by the next step of tracking the DNA helix that locates hemi-methylated sites, is regulated by the conformational state of the protein as a function of ATP binding/hydrolysis. Here, we study how various molecular determinants of a heteroduplex regulate mismatch recognition by MutS, the critical first step of mismatch repair. Using classical DNase I footprinting assays, we demonstrate that the hierarchy of MutS binding to various types of mismatches is identical whether the mismatches are present singly or in multiples. Moreover, this unique hierarchy is indifferent both to the differential level of DNA helical flexibility and to the unpaired status of the mismatched bases in a heteroduplex. Surprisingly, multiple mismatches exhibit reduced affinity of binding to MutS, compared to that of a similar single mismatch. Such a reduction in the affinity might be due to sequence context effects, which we established more directly by studying two identical single mismatches in an altered sequence background. A mismatch, upon simply being flipped at the same location, elicits changes in MutS specific contacts, thereby underscoring the importance of sequence context in modulating MutS binding to mismatches.

Adenosine Triphosphatases↗
PubMed2000

RecA realigns suboptimally paired frames of DNA repeats through a process that requires ATP hydrolysis.

Microsatellite repeats such as mono-, di-, and trinucleotides are highly abundant and viable targets for homologous recombination in the genome. However, if recombination ensues in such repetitive regions, they are intrinsically prone to frame misalignments during pairing and might eventually give rise to genetic instabilities. Suboptimally paired frames lead to an abrogation of branch migration at the junctions of mixed sequences and repeats, due to a heterologous register. If so, can recombination machinery rectify such misalignments in order to avoid subsequent arrest in branch migration? We analyzed Escherichia coli RecA, the universal prototype of a recombinase, for its pairing abilities across repeats. We used a complementary pairing assay to test whether RecA can mediate realignments of stochastically paired suboptimal frames to a maximally aligned register. Here, we demonstrate that RecA-single stranded DNA filament indeed facilitates such a realignment, probably by sliding the paired strands across mono- and di- as well as trinucleotide repeats. These realignments apparently have no net directional bias. Such a putative "motor" function of RecA seems to be ATP hydrolysis-dependent.

Adenosine Triphosphate↗
PubMed2000

ATP-hydrolysis-dependent conformational switch modulates the stability of MutS-mismatch complexes.

The mismatch repair pathway in Escherichia coli has been extensively studied in vitro as well as in vivo. The molecular mechanisms by which nucleotide cofactors regulate the whole process constitute an area of active debate. Here we demonstrate that nucleotide (ADP or ATP) binding to MutS mediates a switch in protein conformation. However, in MutS that is DNA bound, this switch ensues only with ATP and not with ADP and is similar, irrespective of whether it is bound to a homo- or a heteroduplex. The results envisage a minimal model of three confor-mational states of MutS as reflected in: (i) a specific and highly stable MutS-mismatch complex in the absence of a nucleotide; (ii) a specific but less stable complex in the presence of ATP hydrolysis; and (iii) an irreversibly dissociated complex in the presence of ATP binding (ATPgammaS). Such transitions are of relevance to the protein's function in vivo where it has to first recognize a mismatch, followed by a search for hemimethylated sites.

Adenosine Triphosphatases↗
PubMed1999

Fold-back structures at the distal end influence DNA slippage at the proximal end during mononucleotide repeat expansions.

Polymerase slippage during DNA synthesis by the Klenow fragment of DNA polymerase across A, C, G and T repeats (30 bases) has been studied. Within minutes, duplexes that contain only repeats (30 bp) expand dramatically to several hundred base pairs long. Rate comparisons in a repeat duplex when one strand was expanded as against that when both strands were expanded suggest a model of migrating hairpin loops which in the latter case coalesce into a duplex. Moreover, slippage (at the proximal or 3'-end) is subject to positive and negative effects from the 5'-end (distal) of the same strand. Growing T and G strands generate T.A:T and G-G:C motif fold-back structures at the distal end that hamper slippage at the proximal end. On the other hand, growing tails at the distal end upon annealing with excess complementary template accentuates proximal slippage several-fold.

DNA↗
PubMed1999

RecA interacts with Klenow and enhances fidelity of DNA synthesis in vitro.

To understand the molecular basis of RecA-mediated DNA-repair, we tested the replicative fidelity of the large fragment of Pol I (Klenow) in RecA-DNA complexes in vitro. Klenow synthesis was error-prone in naked DNA substrates but essentially error-free in RecA coated complexes. Escherichia coli SSB, causes no such improvement in Klenow fidelity. RecA filaments promote better exonucleolytic proofreading by Klenow than on naked DNA substrates at select sites when replication is "stalled" due to a missing dNTP. Addition of RecA to pyrene sulfonylchloride-labeled Klenow resulted in a specific increase in steady-state fluorescence anisotropy and a concomitant decrease in fluorescence lifetime. These observations suggest the possibility of a direct interaction between RecA and Klenow even in the absence of DNA which may mediate the observed improvement in Klenow fidelity.

Base Sequence↗
PubMed1998

Non-Watson-Crick base pairs modulate homologous alignments in RecA pairing reactions.

Complementary pairing by RecA was examined in vitro to investigate how homology is deciphered from non-homology. Somewhere in a window of 40-50% sequence complementarity, RecA pairing begins to manifest the specificity of homology. Quantitation reveals a hierarchy among non-Watson-Crick mispairs: RecA reaction treats six out of 12 possible mispairs as good ones and three each of the remaining ones as moderate and bad pairs. The mispairs seem to function as independent pairing units free of sequence context effects. The overall strength of pairing is simply the sum of the constituent units. RecA mediated gradation of mispairs, free of sequence context effects, might offer a general thumb-rule for predicting the pairing strength of any alignment that carries multiple mispairs.

Base Composition↗
PubMed1996

Transition metal saccharide chemistry and biology: synthesis, characterization, electrochemistry and EPR studies of oxovanadium (IV) complexes of saccharides and their derivatives and in vitro interaction of some of these with ribonuclease and deoxyribonuclease.

Low molecular weight, water-soluble saccharide complexes of oxovanadium(IV) have been synthesized and characterized by analytical, spectroscopic and electrochemical techniques. All the complexes were found to be mononuclear, possessing the VO2+ moiety. These are shown to be hydrolytically and oxidatively stable over a wide range of pH (1-12) and have been extensively characterized by absorption and EPR spectroscopy and by electrochemistry. Several correlations have been drawn from the data generated. Some of these complexes have been demonstrated to possess in vitro RNase inhibition activity with no effect on DNase. This suggests that these molecules closely mimic the substrate portion of the RNase-catalysed RNA hydrolysis and can act as transition-state analogues to RNase.

Deoxyribonucleases↗
PubMed1995

RecA protein mediates homologous recognition via non-Watson-Crick bonds in base triplets.

E. coli RecA protein, the prototype of a class, forms a helical nucleoprotein filament on single-stranded DNA that recognizes homology in duplex DNA, and initiates the exchange of strands in homologous recombination. The discovery of this reaction some years ago posed a quandary on how a third strand recognizes homology in duplex DNA, whose Watson-Crick bonds face inward in a hydrophobic core of stacked bases. Recent studies have shown that RecA protein promotes homologous recognition via non-Watson-Crick bonds in base triplets. The intermediates in the RecA reaction differ distinctly from triplex DNA that forms non-enzymically. The biological significance of the novel set of DNA interactions by which RecA protein effects homologous recognition is indicated by the importance of this protein in recombination, and the widespread distribution of homologous proteins in prokaryotes and eukaryotes.

Base Composition↗
PubMed1995

How specific is the first recognition step of homologous recombination?

The Escherichia coli RecA protein promotes homologous recognition in base triplets via non-Watson-Crick bonds that differ from those formed nonenzymically from DNA consisting of runs of purines or pyrimidines. Base substitutions reveal recognition to be permissive, consistent with a search for homology that achieves speed at the cost of precision.

Binding Sites↗
PubMed1994

Joints made by RecA protein in the interior of linear duplex DNA: effects of single-stranded ends, length of homology, and dynamic state.

The importance of 3' single-stranded ends in homologous recombination led us to reevaluate reactions of single strands at homologous sites in the interior of linear duplex DNA. As the length of homology increased, the yield of joints increased up to about 2 kb of homology, at which the apparent yields were the same at either 3' or 5' single-stranded ends, or away from ends, although such joints were qualitatively different. In the presence of RecA protein and ATP, joints that formed between any of these single strands and interior sequences in duplex DNA were in a dynamic state in which they constantly recycled. Consequently, their apparent yields at steady state were sensitive to conditions of reaction, such as the concentrations of DNA, salt, and ATP vs ADP, observations which rationalize conflicting reports in the literature. The dynamic state of joints in the interior of duplex DNA strengthens the conclusion [see Burnett et al. (1994)] that RecA protein dissociates joints when it cannot displace the 5' end of a strand from the recipient duplex.

Adenosine Diphosphate↗
PubMed1994

A chimeric Rec-A protein that implicates non-Watson-Crick interactions in homologous pairing.

The helical filament formed by RecA protein on single-stranded DNA plays an important role in homologous recombination and pairs with a complementary single strand or homologous duplex DNA. The RecA nucleoprotein filament also recognizes an identical single strand. The chimeric protein, RecAc38, forms a nucleoprotein filament that recognizes a complementary strand but is defective in recognition of duplex DNA, and is associated with phenotypic defects in repair and recombination. As described here, RecAc38 nucleoprotein filament is also defective in recognition of an identical strand, either when the filament has within it a single strand or duplex DNA. A model that postulates three DNA binding sites rationalizes these observations and suggests that the third binding site mediates non-Watson-Crick interactions that are instrumental in recognition of homology in duplex DNA.

Base Sequence↗
PubMed1994

Formation of base triplets by non-Watson-Crick bonds mediates homologous recognition in RecA recombination filaments.

Whereas complementary strands of DNA recognize one another by forming Watson-Crick base pairs, the way in which RecA protein enables a single strand to recognize homology in duplex DNA has remained unknown. Recent experiments, however, have shown that a single plus strand in the RecA filament can recognize an identical plus strand via bonds that, by definition, are non-Watson-Crick. In experiments reported here, base substitutions had the same qualitative and quantitative effects on the pairing of two identical strands in the RecA filament as on the recognition of duplex DNA by a third strand, indicating that similar non-Watson-Crick interactions govern both reactions.

Base Composition↗
PubMed1994

Resolution of the three-stranded recombination intermediate made by RecA protein. An essential role of ATP hydrolysis.

Previous work has shown that triplex DNA is an intermediate in homologous pairing and strand exchange promoted by RecA protein. Heterology at the proximal end of duplex DNA blocks strand exchange, but triplex joints form nonetheless at the homologous distal end. Experiments on the formation and processing of distal joints revealed that the yield of distal joints depends critically on the concentration of RecA-coated single strands and the adequacy of the ATP-regeneration system, and reflects a steady state. Distal joints reversibly formed and dissociated, as shown by several methods, including a chase with unlabeled duplex DNA. Controls excluded a contribution of exonucleolytic nibbling to the formation of distal joints and the stability of the deproteinized product. RecA protein was bound preferentially by putative triplex sites both in isolated proximal and distal joints. These high affinity sites disappeared from proximal joints as strand exchange progressed, and disappeared from distal joints as the joints dissociated. Dissociation of distal joints under all conditions, however, was completely arrested by the addition of ATP gamma S. Distal triplex joints can be as long as six kilobases. The observed inhibition of the dissociation of such long non-productive triplex intermediates by ATP gamma S leads us to propose that an essential role of ATP hydrolysis in RecA recombinational exchanges may be to ensure that no potentially troublesome triplex DNA remains in the cell.

Adenosine Triphosphate↗