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M M Cox

Publications and source records attributed to M M Cox.

At least 55 records · Page 3Linked to original sources
PubMed1995

Blocked RecA protein-mediated DNA strand exchange reactions are reversed by the RuvA and RuvB proteins.

RecA protein is unable to complete a DNA strand exchange reaction between a circular single-stranded DNA and a linear duplex DNA substrate with heterologous sequences of 375 base pairs at the distal end. Instead, it generates a branched intermediate in which strand exchange has proceeded up to the homology/heterology junction. Addition of the RuvA and RuvB proteins to these stalled intermediates leads to the rapid conversion of intermediates back to the original substrates. The reversal reaction is initiated at the branch, and the hybrid DNA is unwound in the direction opposite to that of the RecA reaction that created it. Under optimal conditions the rate of the reaction exhibits only a modest dependence on the length of hybrid DNA that must be unwound. Products of the reversal reaction are detected within minutes after addition of RuvAB, and appear with an apparent first order progress curve, exhibiting a t1/2 in the range of 6-12 min under optimal conditions. Few molecules that have undergone only partial reversal are detected. This suggests that the assembly or activation of RuvAB on the branched substrate is rate-limiting, while any migration of RuvAB on the DNA to effect unwinding of the hybrid DNA (and reformation of substrate DNA) is very fast. The results are discussed in context of the role of RuvA and RuvB proteins in recombinational DNA repair. We suggest that one function of the RuvAB proteins is to act as an antirecombinase, to eliminate intragenomic crossovers between homologous segments of the bacterial chromosome that might otherwise lead to deleterious inversions or deletions.

Bacterial Proteins↗
PubMed1995

Asymmetry in active complexes of FLP recombinase.

The FLP recombinase promotes a site-specific recombination reaction in the 2mu plasmid of yeast. The protein-DNA complex that carries out the reaction is asymmetric. Three FLP monomers bound to specific FLP-recognition sequences are required to efficiently carry out one set of reciprocal DNA cleavage and strand exchange events on a Holliday junction substrate. If a fourth monomer plays an auxiliary role in the reaction, it is bound without sequence specificity. The data suggest a modified model for cleavage of DNA in trans by the FLP recombinase that might help reconcile some seemingly conflicting resulted obtained with integrase class recombinases.

Base Sequence↗
PubMed1995

RuvB protein-mediated ATP hydrolysis: functional asymmetry in the RuvB hexamer.

A survey of RuvB protein-mediated ATP hydrolysis yields the following observations. (1) The RuvB protein exhibits a DNA-independent ATPase activity with a turnover number (based on a RuvB monomer) approaching 6 min-1 and a Km of 154 microM. Single-stranded DNA and linear duplex DNA have small but significant effects on this activity. (2) At ATP concentrations near the Km, the ATPase activity is attenuated after approximately 60 turnovers/RuvB monomer. The attenuation does not reflect inhibition by ADP. Addition of ATP to 3 mM triggers an immediate resumption of ATP hydrolysis. The attention is enhanced somewhat by ssDNA and reduced somewhat by linear dsDNA. (3) ATP hydrolysis is dramatically stimulated by circular dsDNA, reinforcing the notion that RuvB translocates along the DNA in a reaction coupled to ATP hydrolysis. The kcat increases by at least 2-4-fold on circular duplexes depending on conditions, and the inactivation of RuvB at ATP concentrations near the Km does not occur. The ATPase activity on circular dsDNA also exhibits a partial substrate inhibition by ATP. (4) Optimal ATP hydrolysis requires approximately 1 DNA circle/RuvB hexamer, suggesting that multiple RuvB hexamers on a circle have an inhibitory effect on the ATPase activity. (5) With or without any of these DNA cofactors, a burst of ATP hydrolysis is observed under pre-steady-state conditions equivalent to 1 ATP per 3-3.3 RuvB monomers (2 ATP/hexamer). The substrate inhibition and burst results suggest the presence of nonequivalent ATP hydrolytic sites in a RuvB hexamer. The attenuation of ATPase activity observed under some conditions may also be a manifestation of nonequivalent ATP hydrolytic sites.

Adenosine Diphosphate↗
PubMed1995

Occurrence of three-stranded DNA within a RecA protein filament.

A RecA protein-generated triple-stranded DNA species can be observed by electron microscopy, within narrowly defined conditions. Three-stranded DNA is detected only when initiation of normal DNA strand exchange is precluded by heterologous sequences within the duplex DNA substrate, when ATP is hydrolyzed, and when the DNA is cross-linked with a psoralen derivative prior to removal of RecA filaments. When adenosine 5'-O-(thiotriphosphate) is used, only the product hybrid duplex DNA can be cross-linked within the RecA filament. The third strand is either displaced or interwound in a conformation that does not permit cross-linking. When ATP is hydrolyzed by RecA, all three strands are cross-linked within the filament in a complex pattern that suggests a dynamic structure. This structure is altered when RecA protein is removed before cross-linking. Hsieh et al. (1990) and Rao et al. (1991, 1993) have proposed, on the basis of nuclease protection and chemical modification studies, that a stable triple-stranded DNA species can persist after removal of RecA protein. We have been unable to visualize these triple-stranded structures by the methods used in the present investigation. When RecA removal was followed immediately by interstrand cross-linking, only the two strands of the hybrid duplex DNA were cross-linked.

Adenosine Triphosphate↗
PubMed1995

Transient DNA breaks associated with programmed genomic deletion events in conjugating cells of Tetrahymena thermophila.

Thousands of programmed genomic deletion events occur during macronuclear development in Tetrahymena thermophila. Two of the deleted segments, called M and R, have been particularly well-characterized. Using ligation-mediated PCR, we have detected DNA strand breaks that correlate temporally and structurally with the deletion events in the M and R regions. The ends appear at positions that correspond precisely to boundaries of deleted sequences, as defined by observed chromosomal junctions found after deletion is complete. They occur exclusively during the known DNA rearrangement period in macronuclear development. The breaks are staggered by 4 bp in the complementary strands. Several alternative breaks were found at the end of one deleted region, consistent with multiple alternative chromosomal junctions detected previously. The free 5' ends generated at the breaks are phosphorylated. A purine residue always occurs at the free 3' ends, with an adenosine appearing in 11 of 12 cases. Patterns found in the detected break sites suggest rules that define the ends of the deleted segments within a transposon-like deletion mechanism.

Animals↗
PubMed1995

The deduced Vibrio cholerae RecA amino-acid sequence.

The nucleotide sequence of the recA gene of Vibrio cholerae (Vc) has been determined. The amino acid (aa) sequence of the protein product is very similar to other known RecA aa sequences. However, this sequence does not agree with a previously reported Vc RecA aa sequence [Ghosh et al., Nucleic Acids Res. 20 (1992) 372].

Amino Acid Sequence↗
PubMed1994

The fate of deleted DNA produced during programmed genomic deletion events in Tetrahymena thermophila.

Thousands of DNA deletion events occur during macronuclear development in the ciliate Tetrahymena thermophila. In two deleted genomic regions, designated M and R, the eliminated sequences form circles that can be detected by PCR. However, the circles are not normal products of the reaction pathway. The circular forms occur at very low levels in conjugating cells, but are stable. Sequencing analysis showed that many of the circles (as many as 50% of those examined) reflected a precise deletion in the M and R regions. The remaining circles were either smaller or larger and contained varying lengths of sequences derived from the chromosomal DNA surrounding the eliminated region. The chromosomal junctions left behind after deletion were more precise, although deletions in either the M or R regions can generate any of several alternative junctions (1). Some new chromosomal junctions were detected in the present study. The results suggest that the deleted segment is released as a linear DNA species that is degraded rapidly. The species is only rarely converted to the stable circles we detect. The deletion mechanism is different from those proposed for deletion events in hypotrichous ciliates (2-4), and does not reflect a conservative site-specific recombination process such as that promoted by the bacteriophage lambda integrase (5).

Animals↗
PubMed1994

RuvA and RuvB proteins facilitate the bypass of heterologous DNA insertions during RecA protein-mediated DNA strand exchange.

RecA protein-mediated DNA strand exchange between circular single-stranded DNA and linear duplex DNA readily bypasses short (up to 100 base pairs) heterologous inserts in one of the DNA substrates. Larger heterologous inserts are bypassed with decreasing efficiency, and inserts larger than 200 base pairs substantially block RecA-mediated DNA strand exchange. The RuvA and RuvB proteins dramatically facilitate the bypass of larger heterologous inserts. When the RuvA and RuvB proteins are added to an ongoing RecA protein-mediated strand exchange reaction, interior heterologous inserts of 1 kilobase pair are bypassed at significant frequencies. The RuvA, RuvB, and RecA proteins are all required for this activity. Bypass occurs only when homologous sequences are present on both sides of the insert. When the heterologous insert is positioned at either end of the linear duplex substrate, the RuvA and RuvB proteins do not significantly increase product formation in RecA protein-mediated DNA strand exchange reactions. The results suggest an important role for RuvA and RuvB in the bypass of DNA structural barriers during recombinational DNA repair.

Adenosine Triphosphate↗
PubMed1994

On the role of ATP hydrolysis in RecA protein-mediated DNA strand exchange. III. Unidirectional branch migration and extensive hybrid DNA formation.

We have identified two functions for RecA-mediated ATP hydrolysis during DNA strand exchange. First, ATP hydrolysis renders RecA protein-mediated DNA strand exchange unidirectional (5' to 3' with respect to the single-stranded DNA). In the presence of a nonhydrolyzable analog adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), DNA strand exchange is bidirectional. Second, ATP hydrolysis is required for extensive formation of hybrid DNA. In the presence of ATP hydrolysis, the length of the exchanged region is limited only by the available homology, whereas in the absence of ATP hydrolysis, only 2 kilobase pairs or less of hybrid DNA are formed before branch migration is blocked in the majority of paired intermediates. Both of these functions of RecA protein-mediated ATP hydrolysis are crucial in ensuring the effectiveness of recombinational DNA repair, especially when the lesion to be repaired is distant from the initial crossover point.

Adenosine Triphosphate↗
PubMed1994

Future evaluation of antiarrhythmic therapy.

The expansion of antiarrhythmic therapy beyond pharmacologic agents to include surgery, devices, and ablation procedures, plus the reaffirmation by the Cardiac Arrhythmia Suppression Trial (CAST) of the need for concurrent placebo-controlled trials to establish a mortality benefit, have resulted in the need to consider the requirements for evaluating therapy. Pharmacologic therapy may be used in three ways: (1) primary; (2) alternative; and (3) adjunctive. To accurately identify a mortality benefit from primary therapy, a placebo-controlled study is necessary. In contrast, control of symptoms may be identified without the same rigorous demands. Current data are limited by the absence of true negative controls for most interventions that claim a possible mortality benefit. Alternative therapy provides a choice between equally effective therapies, neither of which has necessarily been documented to have a mortality benefit. Adjunctive therapy is that which is used for control of symptoms, whereas another therapy is used to provide a presumed or proved mortality benefit. For any of these approaches, therapy must be further evaluated in terms of four modifying variables: (1) impact of therapy on the basis of both its efficacy and efficiency; (2) interpretation of outcome data based on analysis of competing risks; (3) measurement of efficacy in terms of extension of life; and (4) analysis of outcome as the equilibrium between antiarrhythmic benefit and proarrhythmic risk. With these approaches a rational analysis of the effect of therapy and its cost-based benefit can be achieved.

Anti-Arrhythmia Agents↗
PubMed1994

Why does RecA protein hydrolyse ATP?

RecA is a DNA-dependent ATPase involved in DNA-strand repair. Most of the ATP hydrolysis that occurs in a RecA nucleoprotein filament is implicitly considered to be irrelevant in many current models for RecA-mediated DNA-strand exchange. However, preventing RecA from hydrolysing ATP alters its behavior, suggesting that ATP hydrolysis by RecA is more than incidental. This review explores recent results detailing the effects and rates of ATP hydrolysis by RecA, and models are proposed that permit us to account quantitatively for ATP consumption by this protein.

Adenosine Triphosphate↗
PubMed1993

A reverse DNA strand exchange mediated by recA protein and exonuclease I. The generation of apparent DNA strand breaks by recA protein is explained.

The combined action of exonuclease I and recA protein leads to a kind of reverse DNA strand exchange in which joint molecules formed on the "wrong" or distal end of a linear duplex in the presence of ATP are stabilized by exonuclease I degradation of the displaced (+) strand. Continued pairing and degradation of the displaced strand leads to strand exchange that appears to progress with a polarity opposite that of the normal recA protein promoted reaction (i.e. 3'-5' with respect to the (+) strand). However, in contrast to the normal 5'-3' strand exchange, the displaced strand is completely degraded in the process. When the linear duplex DNA substrate has a heterologous region at the 5' (proximal) end, the major product (described in a previous study (Bedale, W. A., Inman, R. B., and Cox, M. M. (1991) J. Biol. Chem. 266, 6499-6510)) is a circular duplex DNA molecule with a double-stranded tail whose length corresponds closely to the heterologous segment of the substrate. The origin of this product is here shown to be the result of the exonuclease activity of exonuclease I (either added exogenously or present as a trace contaminant of recA protein or SSB protein preparations), as opposed to endonucleolytic or mechanical breakage. The levels of exonuclease I required to generate these products are sufficiently low that they are undetected by assays for exonuclease contamination in recA protein preparations. These results demonstrate that the interplay of recA protein with other enzymes can have a profound effect on both the mechanism and outcome of recA protein-promoted DNA strand exchange. They also demonstrate that the (+) strand of the duplex DNA substrate is at least transiently displaced in recA protein-mediated pairing even when joint molecules are limited to the distal end.

Bacteriophage M13↗
PubMed1993

A shared pathway in atrioventricular nodal reentrant tachycardia and atrial flutter: implications for pathophysiology and therapy.

Atrioventricular (AV) nodal reentrant tachycardia and atrial flutter are considered 2 distinct supraventricular tachycardias. Recent clinical and experimental data suggest that both these tachycardias include an area in the lower right atrial septum in their reentrant pathways. This study was designed to test the hypothesis that there is an association between the mechanisms of AV nodal reentrant tachycardia and atrial flutter because of a shared pathway of reentry. Consecutive patients referred for evaluation and management of supraventricular tachycardia, thought to be due to AV nodal reentry, underwent electrophysiologic testing protocols designed to induce both AV nodal reentrant tachycardia and atrial flutter, if present. Fifteen of 29 patients (52%) had both AV nodal reentrant tachycardia and atrial flutter induced during electrophysiologic testing. Seven of these 15 patients (47%) underwent transcatheter radiofrequency current application (mean power 34 +/- 4 W) against the tricuspid annulus above the coronary sinus. In each patient, neither AV nodal reentrant tachycardia nor atrial flutter could be induced after the procedure. Repeat study after successful ablation (mean 6 days) showed no inducible supraventricular arrhythmia of either type at baseline study or during isoproterenol infusion. Atrial flutter occurs frequently (15 of 29 patients; 52%) in patients with AV nodal reentrant tachycardia, because of a shared pathway in their reentry circuits. Because of this shared pathway, both arrhythmias can be ablated at the same site. These observations promote new insights into the mechanism and therapeutics of supraventricular tachycardias.

Adult↗
PubMed1993

Relating biochemistry to biology: how the recombinational repair function of RecA protein is manifested in its molecular properties.

The multiple activities of the RecA protein in DNA metabolism have inspired over a decade of research in dozens of laboratories around the world. This effort has nevertheless failed to yield an understanding of the mechanism of several RecA protein-mediated processes, the DNA strand exchange reactions prominent among them. The major factors impeding progress are the invalid constraints placed upon the problem by attempting to understand RecA protein-mediated DNA strand exchange within the context of an inappropriate biological paradigm-namely, homologous genetic recombination as a mechanism for generating genetic diversity. In this essay I summarize genetic and biochemical data demonstrating that RecA protein evolved as the central component of a recombinational DNA repair system, with the generation of genetic diversity being a sometimes useful byproduct, and review the major in vitro activities of RecA protein from a repair perspective. While models proposed for both recombination and recombinational repair often make use of DNA strand cleavage and transfer steps that appear to be quite similar, the molecular and thermodynamic requirements of the two processes are very different. The recombinational repair function provides a much more logical and informative framework for thinking about the biochemical properties of RecA and the strand exchange reactions it facilitates.

Adenosine Triphosphate↗
PubMed1993

Alternating Wenckebach periods and allied arrhythmias.

Alternating Wenckebach periods (AWPs) are episodes of 2:1 block during which the PR, AH, or AV intervals of the conducted beats gradually increase until a greater degree of block ensues. Most episodes occur at the AV node, but some have also been reported in other structures. AWPs are usually attributed to multilevel block due to transverse (horizontal) dissociation. This assumption was initially based on a method in which the solutions to difficult electrocardiographic rhythms were arrived at by analysis and deduction based on the knowledge existing at that particular time. Subsequently, it was reinforced by information extrapolated from intracardiac recordings performed in patients with documented multilevel block in separate anatomical structures (atria, AV node, and His bundle), as well as from microelectrode studies and computer simulations. Although AWPs are frequently observed in clinical tracings, those occurring at the AV node are best categorized during incremental atrial stimulation because then they occupy a specific point in the wide spectrum of tachycardia dependent AV nodal conduction disturbances. In fact, the A:H ratios occurring in the episodes where the degree of block increases can be represented by "universal" mathematical formulas. However, in the clinical setting, drugs affecting the electrophysiology of the node can alter the pacing induced symmetry by producing additional differential effects on the various levels. The latter still requires further elucidation.

Atrioventricular Node↗
PubMed1992

On the role of ATP hydrolysis in RecA protein-mediated DNA strand exchange. I. Bypassing a short heterologous insert in one DNA substrate.

RecA protein promotes a substantial DNA strand exchange reaction in the presence of adenosine 5'-O-3-(thio)triphosphate (ATP gamma S) (Menetski, J.P., Bear, D.G., and Kowalczykowski, S.C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 21-25), calling into question the role of ATP hydrolysis in the strand exchange reaction. Here, we demonstrate that the ATP gamma S-mediated reaction can go to completion when the duplex DNA substrate is only 1.3 kilobase pairs in length. The ATP gamma S-mediated reaction, however, is completely blocked by a 52-base pair heterologous insertion in either DNA substrate. This same barrier is readily bypassed when ATP replaces ATP gamma S. This indicates that at least one function of recA-mediated ATP hydrolysis is to bypass structural barriers in one or both DNA substrates during strand exchange. This suggests that ATP hydrolysis is directly coupled to the branch migration phase of strand exchange, not to promote strand exchange between homologous DNA substrates during recombination, but instead to facilitate the bypass of structural barriers likely to be encountered during recombinational DNA repair.

Adenosine Triphosphate↗
PubMed1992

On the role of ATP hydrolysis in RecA protein-mediated DNA strand exchange. II. Four-strand exchanges.

RecA protein promotes a substantial DNA strand exchange reaction in the presence of adenosine 5'-O-3-(thio)triphosphate (ATP gamma S) (Menetski et al., 1990), calling into question the role of ATP hydrolysis in this reaction. We demonstrate here that the ATP gamma S-mediated process is restricted to homologous strand exchange reactions involving three strands. In four-strand exchanges between a gapped duplex circle and a second linear duplex, joint molecules are formed in the gap but are not extended into the four-strand region when ATP gamma S is present. This result provides evidence that one function of ATP hydrolysis in the recA system is to facilitate reciprocal DNA strand exchange involving four strands. Implications with respect to the role of four-stranded pairing intermediates and the mechanistic relationship between three- and four-strand exchange reactions are discussed.

Adenosine Triphosphate↗