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Biomedical subjects

M M Cox

Publications and source records attributed to M M Cox.

At least 37 records · Page 2Linked to original sources
PubMed1997

Recombinational DNA repair: the RecF and RecR proteins limit the extension of RecA filaments beyond single-strand DNA gaps.

In the presence of both the RecF and RecR proteins, RecA filament extension from a single strand gap into adjoining duplex DNA is attenuated. RecR protein alone has no effect, and RecF protein alone has a reduced activity. The RecFR complexes bind randomly, primarily to the duplex regions of the DNA, and the extension of the RecA filament is halted at the first complex encountered. A very slow lengthening of RecA filaments observed in the presence of RecFR is virtually eliminated when RecF is replaced with an RecF mutant protein that does not hydrolyze ATP. These observations are incorporated into an expanded model for the functions of RecF, RecO, and RecR proteins in the early stages of postreplication DNA repair.

Bacterial Proteins↗
PubMed1997

RecA as a motor protein. Testing models for the role of ATP hydrolysis in DNA strand exchange.

ATP hydrolysis (by RecA protein) fundamentally alters the properties of RecA protein-mediated DNA strand exchange reactions. ATP hydrolysis renders DNA strand exchange unidirectional, greatly increases the lengths of hybrid DNA created, permits the bypass of heterologous DNA insertions in one or both DNA substrates, and is absolutely required for exchange reactions involving four DNA strands. There are at least two viable models to explain how ATP hydrolysis is coupled to DNA strand exchange so as to bring about these effects. The first couples ATP hydrolysis to a redistribution of RecA monomers within a RecA filament. The second couples ATP hydrolysis to a facilitated rotation of the DNA substrates. The RecA monomer redistribution model makes the prediction that heterology bypass should not occur if the single-stranded DNA substrate is linear. The facilitated DNA rotation model predicts that RecA protein should promote the separation of paired DNA strands within a RecA filament if one of them is contiguous with a length of DNA being rotated about the filament exterior. Here, a facile bypass of heterologous insertions with linear DNA substrates is demonstrated, providing evidence against a role for RecA monomer redistribution in heterology bypass. In addition, we demonstrate that following a four-strand DNA exchange reaction, a distal segment of DNA hundreds of base pairs in length can be unwound in a nonreciprocal phase of the reaction, consistent with the direct coupling of an ATP hydrolytic motor to the proposed DNA rotation.

Adenosine Triphosphate↗
PubMed1997

RecA filament dynamics during DNA strand exchange reactions.

The role of ATP hydrolysis in RecA protein-mediated DNA strand exchange reactions remains controversial. Competing models suggest that ATP hydrolysis is coupled either to a simple redistribution of RecA monomers within a filament to repair filament discontinuities, or more directly to rotation of the DNA substrates to drive branch movement unidirectionally. Here, we test key predictions of the RecA redistribution idea. When ATP is hydrolyzed, DNA strand exchange is accompanied by a RecA exchange reaction, between free and bound RecA protomers in the interior of RecA filaments, that meets a central prediction of the model. The RecA protomer exchange is not required for, and does not occur during, the "search for homology" in which the single-stranded DNA within a RecA-ssDNA nucleoprotein filament is homologously aligned with the duplex DNA. Instead, the RecA exchange is triggered by the completion of strand exchange (a strand switch to generate a hybrid DNA product) in any given segment of the filament. In effect, formation of hybrid DNA leads to a change in filament conformation to one with properties approximating those of RecA filaments bound to double-stranded DNA. Addition of the RecA K72R mutant protein to a reaction with the wild type protein leads to the formation of mixed filaments and a poisoning of the DNA strand exchange reaction. Under some conditions, a facile RecA protomer exchange is observed, and significant ATP is hydrolyzed, even though DNA strand exchange is entirely blocked by the mutant protein. A redistribution of RecA protomers coupled to ATP hydrolysis is not sufficient in itself to explain how ATP hydrolysis facilitates DNA strand exchange. A RecA protomer exchange may nevertheless play an important role in the DNA strand exchange process.

Adenosine Triphosphate↗
PubMed1997

RecA protein filaments: end-dependent dissociation from ssDNA and stabilization by RecO and RecR proteins.

RecA protein filaments formed on circular (ssDNA) in the presence of ssDNA binding protein (SSB) are generally stable as long as ATP is regenerated. On linear ssDNA, stable RecA filaments are believed to be formed by nucleation at random sites on the DNA followed by filament extension in the 5' to 3' direction. This view must now be enlarged as we demonstrate that RecA filaments formed on linear ssDNA are subject to a previously undetected end-dependent disassembly process. RecA protein slowly dissociates from one filament end and is replaced by SSB. The results are most consistent with disassembly from the filament end nearest the 5' end of the DNA. The bound SSB prevents re-formation of the RecA filaments, rendering the dissociation largely irreversible. The dissociation requires ATP hydrolysis. Disassembly is not observed when the pH is lowered to 6.3 or when dATP replaces ATP. Disassembly is not observed even with ATP when both the RecO and RecR proteins are present in the initial reaction mixture. When the RecO and RecR proteins are added after most of the RecA protein has already dissociated, RecA protein filaments re-form after a short lag. The newly formed filaments contain an amount of RecA protein and exhibit an ATP hydrolysis rate comparable to that observed when the RecO and RecR proteins are included in the initial reaction mixture. The RecO and RecR proteins thereby stabilize RecA filaments even at the 5' ends of ssDNA, a fact which should affect the recombination potential of 5' ends relative to 3' ends. The location and length of RecA filaments involved in recombinational DNA repair is dictated by both the assembly and disassembly processes, as well as by the presence or absence of a variety of other proteins that can modulate either process.

Bacterial Proteins↗
PubMed1997

Convenient and reversible site-specific targeting of exogenous DNA into a bacterial chromosome by use of the FLP recombinase: the FLIRT system.

We have created a system that utilizes the FLP recombinase of yeast to introduce exogenous cloned DNA reversibly at defined locations in the Escherichia coli chromosome. Recombination target (FRT) sites can be introduced permanently at random locations in the chromosome on a modified Tn5 transposon, now designed so that the inserted FRT can be detected and its location mapped with base pair resolution. FLP recombinase is provided as needed through the regulated expression of its gene on a plasmid. Exogenous DNA is introduced on a cloning vector that contains an FRT, selectable markers, and a replication origin designed to be deleted prior to electroporation for targeting purposes. High yields of targeted integrants are obtained, even in a recA background. This system permits rapid and precise excision of the introduced DNA when needed, without destroying the cells. The efficiency of targeting appears to be affected only modestly by transcription initiation upstream of the chromosomal FRT site. With rare exceptions, FRTs introduced to the bacterial chromosome are targeted with high efficiency regardless of their location. The system should facilitate studies of bacterial genome structure and function, simplify a wide range of chromosomal cloning applications, and generally enhance the utility of E. coli as an experimental organism in biotechnology.

Blotting, Southern↗
PubMed1996

Characterization of a mutant RecA protein that facilitates homologous genetic recombination but not recombinational DNA repair: RecA423.

A recA mutant (recA423; Arg169-->His), with properties that should help clarify the relationship between the biochemical properties of RecA protein and its two major functions, homologous genetic recombination and recombinational DNA repair, has been isolated. The mutant has been characterized in vivo and the purified RecA423 protein has been studied in vitro. The recA423 cells are nearly as proficient in conjugational recombination, transductional recombination, and recombination of lambda red- gam- phage as wild-type cells. At the same time, the mutant cells are deficient for intra-chromosomal recombination and nearly as sensitive to UV irradiation as a recA deletion strain. The cells are proficient in SOS induction, and results indicate the defect involves the capacity of RecA protein to participate directly in recombinational DNA repair. In vitro, the RecA423 protein binds to single-stranded DNA slowly, with an associated decline in the ATP hydrolytic activity. The RecA423 protein promoted a limited DNA strand exchange reaction when the DNA substrates were homologous, but no bypass of a short heterologous insert in the duplex DNA substrate was observed. These results indicate that poor binding to DNA and low ATP hydrolysis activity can selectively compromise certain functions of RecA protein. The RecA423 protein can promote recombination between homologous DNAs during Hfr crosses, indicating that the biochemical requirements for such genetic exchanges are minimal. However, the deficiencies in recombinational DNA repair suggest that the biochemical requirements for this function are more exacting.

Adenosine Triphosphatases↗
PubMed1996

Allosteric effects of RuvA protein, ATP, and DNA on RuvB protein-mediated ATP hydrolysis.

A detailed characterization of RuvB protein-mediated ATP hydrolysis in the presence of RuvA protein has provided (a) the steady-state kinetic parameters of ATP hydrolysis within a RuvAB complex and (b) several insights into the mechanism of ATP hydrolysis and its coupling to translocation on DNA. In general, the RuvA protein increases the kcat and decreases the Km for the RuvB ATPase activity. DNA has a much greater effect on the kinetics of ATP hydrolysis when RuvA is present, consistent with a role of RuvA in facilitating the interaction between RuvB and DNA. Mechanistic clues come from deviations from normal steady-state kinetic behavior. A previously described burst of ATP hydrolysis, corresponding to two ATPs per RuvB hexamer [Marrione & Cox (1995) Biochemistry 34, 9809-9818], is still observed in the presence of RuvA protein. This suggests a functional asymmetry in the RuvB hexamer. There is a gradual attenuation of ATP hydrolysis when RuvB protein, alone or in the presence of RuvA protein, hydrolyzes ATP at ATP concentrations below the Km. The attenuation is observed even though an ATP regeneration system is present. ATP hydrolysis simply halts after a limited number of turnovers. The attenuation is reversible, and the effects of RuvA protein, DNA, and additional ATP in reversing the effect provide evidence for a complex array of allosteric interactions operating within the RuvB hexameric helicase. We propose a model in which individual subunits in a RuvB hexamer are functionally paired, with the three pairs moving sequentially and cooperatively through a multistep ATP hydrolytic cycle.

Adenosine Triphosphatases↗
PubMed1996

Developmentally programmed DNA deletion in Tetrahymena thermophila by a transposition-like reaction pathway.

We provide a molecular description of key intermediates in the deletion of two internal eliminated sequences (IES elements), the M and R regions, during macronuclear development in Tetrahymena thermophila. Using a variety of PCR-based methods in vivo, double-strand breaks are detected that are generated by hydrolytic cleavage and correspond closely to the observed chromosomal junctions left behind in the macronuclei. The breaks exhibit a temporal and structural relationship to the deletion reaction that provides strong evidence that they are intermediates in the deletion pathway. Breaks in the individual strands are staggered by 4 bp, producing a four nucleotide 5' extension. Evidence is presented that breaks do not occur simultaneously at both ends. The results are most consistent with a deletion mechanism featuring initiation by double-strand cleavage at one end of the deleted element, followed by transesterification to generate the macronuclear junction on one DNA strand. An adenosine residue is found at all the nucleophilic 3' ends used in the postulated transesterification step. Evidence for the transesterification step is provided by detection of a 3' hydroxyl that would be liberated by such a step at a deletion boundary where no other DNA strand ends are detected.

Animals↗
PubMed1996

RecA protein dynamics in the interior of RecA nucleoprotein filaments.

We characterize aspects of the conformation and dynamic state of RecA filaments when bound to dsDNA that are specifically linked to the presence of the second of the two bound DNA strands. Filaments bound to dsDNA exhibit a facile exchange between free and bound RecA monomers or oligomers in the filament interior that is not seen on ssDNA. The RecA mutant K72R, which binds but does not hydrolyze ATP, forms mixed filaments with wild type RecA protein under some conditions. In the presence of dATP, mixed filaments are formed on dsDNA or ssDNA in which the RecA K72R content approximately reflects the proportion of the K72R mutant in the total RecA protein present when the filament is formed. In the presence of ATP, mixed filaments are formed on dsDNA, but the mutant protein strongly inhibits the binding of wtRecA protein to single-stranded DNA. When RecA K72R is added to pre-formed filaments containing only wild-type RecA protein on single-stranded DNA, little of the mutant protein exchanges into the filament. Exchange occurs readily, however, when the filament is bound to double-stranded DNA. The presence of a second DNA strand in RecA-dsDNA filaments produces as altered and more dynamic filament state relative to filaments formed on single-stranded DNA. The results point to a substantial alteration in filament state when synapsis occurs during RecA protein-mediated DNA strand exchange.

Adenosine Triphosphate↗
PubMed1996

DNA strand exchange promoted by RecA K72R. Two reaction phases with different Mg2+ requirements.

Replacement of lysine 72 in RecA protein with arginine produces a mutant protein that binds but does not hydrolyze ATP. The protein nevertheless promotes DNA strand exchange (Rehrauer, W. M., and Kowalczykowski, S. C. (1993) J. Biol. Chem. 268, 1292-1297). With RecA K72R protein, the formation of the hybrid DNA product of strand exchange is greatly affected by the concentration of Mg2+ in ways that reflect the concentration of a Mg.dATP complex. When Mg2+ is present at concentrations just sufficient to form the Mg.dATP complex, substantial generation of completed product hybrid DNAs over 7 kilobase pairs in length is observed (albeit slowly). Higher levels of Mg2+ are required for optimal uptake of substrate duplex DNA into the nucleoprotein filament, indicating that the formation of joint molecules is facilitated by Mg2+ levels that inhibit the subsequent migration of a DNA branch. We also show that the strand exchange reaction promoted by RecA K72R, regardless of the Mg2+ concentration, is bidirectional and incapable of bypassing structural barriers in the DNA or accommodating four DNA strands. The reaction exhibits the same limitations as that promoted by wild type RecA protein in the presence of adenosine 5'-O-(3-thio)triphosphate. The Mg2+ effects, the limitations of RecA-mediated DNA strand exchange in the absence of ATP hydrolysis, and unusual DNA structures observed by electron microscopy in some experiments, are interpreted in the context of a model in which a fast phase of DNA strand exchange produces a discontinuous three-stranded DNA pairing intermediate, followed by a slow phase in which the discontinuities are resolved. The mutant protein also facilitates the autocatalytic cleavage of the LexA repressor, but at a reduced rate.

Arginine↗
PubMed1995

An interaction between the Escherichia coli RecF and RecR proteins dependent on ATP and double-stranded DNA.

The DNA binding and ATPase activities of RecF protein are modulated by RecR protein. Stoichiometric amounts of RecF protein bind to double-stranded (ds) DNA (about 1 RecF monomer/4-6 base pairs) in the presence of adenosine 5'-O-(3-thio)triphosphate (ATP gamma S), forming a homogeneous protein coating on the DNA. Little or no cooperativity is evident in the binding process. In the presence of ATP, RecF binding to dsDNA is much weaker, and no RecF protein coating forms. Instead, small numbers of RecF protomers are interspersed randomly along the DNA. RecR protein does not bind appreciably to the dsDNA under these same conditions. However, a protein coating, similar to that which was observed with RecF protein alone in the presence of ATP gamma S, was produced when both RecF and RecR proteins were incubated with dsDNA in the presence of ATP. An interaction between RecF and RecR enables both proteins to bind tightly to the dsDNA in an approximately 1:1 molar ratio. We also report a weak ATP hydrolytic activity of RecF which is stimulated by RecR.

Adenosine Triphosphatases↗
PubMed1995

Acute and long-term beta-adrenergic blockade for patients with neurocardiogenic syncope.

OBJECTIVES: This study was designed to prospectively evaluate the long-term outcome of drug therapy guided by head-up tilt testing for the management of unexplained syncope and near syncope. BACKGROUND: Head-up tilt testing is used to evaluate patients with unexplained syncope. The validity of acute drug testing and the efficacy of long-term oral therapy for prevention of recurrent syncope have not been investigated in large patient groups. METHODS: We studied 296 consecutive patients with unexplained syncope or near syncope who underwent 80 degrees head-up tilt testing with and without isoproterenol challenge. The efficacy of intravenous and oral beta-blocker therapy was evaluated by repeat testing. Patients with both positive and negative responses to therapy were followed up for rates of recurrence of syncope. RESULTS: A total of 193 patients (65%) had a positive tilt test response; 89% of these 193 required isoproterenol challenge to elicit this response. Patients with a positive tilt test result had lower values for heart rate at rest (mean +/- SD 69 +/- 13 vs. 74 +/- 14 beats/min, p = 0.046) and systolic blood pressure (137 +/- 28 vs. 145 +/- 30 mm Hg, p = 0.0018) at baseline than did the patients with a negative tilt test result. Intravenous propranolol blocked the positive response in 163 (90%) of 181 patients retested. Oral beta-blockers were effective by tilt test criteria in 118 (94%) of 125 patients; 12 (10%) had recurrent clinical symptoms while taking beta-blockers. Eight (42%) of 19 patients who had a negative tilt test response during beta-blocker therapy had recurrent symptoms when they stopped therapy. Three (23%) of 13 patients receiving empiric beta-blocker therapy had recurrent symptoms. The follow-up period for the patients with a positive tilt test result was 28 +/- 11 months (range 5 to 48). CONCLUSIONS: Intravenous propranolol is effective in preventing neurocardiogenic syncope diagnosed during head-up tilt testing and predicts the response to oral beta-blocker therapy. Oral beta-blocker therapy prevents recurrent syncope in the majority of patients. Recurrence of syncope is lowest when efficacy of oral beta-blocker therapy is confirmed by repeat head-up tilt testing.

Administration, Oral↗
PubMed1995

A protein dissociation step limits turnover in FLP recombinase-mediated site-specific recombination.

When two ongoing FLP-mediated recombination reactions are mixed, formation of cross-products is subject to a lag of several minutes, and the subsequent rate of cross-product formation is greatly reduced relative to normal reaction progress curves. The lag reflects the formation of a stable complex containing multiple FLP monomers and two FLP recombination target-containing DNA recombination products, a process completed within 5-10 min after addition of FLP recombinase to a reaction mixture. The reaction products are sequestered within this complex for an extended period of time, unavailable for further reaction. The length of the lag increases with increasing FLP protein concentration and is not affected by the introduction of unreacted (non FLP-bound) substrate. The results provide evidence that disassembly of FLP complexes from products occurs in a minimum of two steps. At least one FLP protein monomer is released from reaction complexes in a discrete step that leaves the reaction products sequestered. The recombination products are released in a form free to react with other FLP recombination target-containing DNA molecules only after at least one additional dissassembly step. One or both of these disassembly steps are rate limiting for reaction turnover under conditions often used to monitor FLP-mediated recombination in vitro.

Base Sequence↗
PubMed1995

Quantitative RecA protein binding to the hybrid duplex product of DNA strand exchange.

Following a DNA strand exchange reaction, RecA protein remains bound to the hybrid DNA product. DNA strand exchange reactions were carried out under optimal conditions in the presence of both RecA protein and SSB protein. As monitored by a sensitive DNA underwinding assay, all of the RecA protein present in the RecA nucleoprotein filament that initiates the strand exchange reaction can be accounted for on the hybrid DNA. As shown elsewhere, the SSB is bound to the displaced single DNA strand. Previous studies showed that RecA protein will dissociate from dsDNA when ADP levels build up, or transfer from dsDNA to ssDNA when the latter is not bound by SSB. The present work (done with ATP regeneration and SSB) shows that efficient strand exchange occurs in the absence of a net dissociation or transfer of RecA monomers from the filament. Such a dissociation or transfer is therefore not a mechanistic requirement for DNA strand exchange. The results provide evidence against some models proposed for the DNA strand exchange mechanism.

Adenosine Triphosphate↗