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M M Cox

Publications and source records attributed to M M Cox.

At least 109 records · Page 6Linked to original sources
PubMed1987

Stable binding of recA protein to duplex DNA. Unraveling a paradox.

recA protein binding to duplex DNA is a complicated, multistep process. The final product of this process is a stably bound complex of recA protein and extensively unwound double-stranded DNA. recA monomers within the complex hydrolyze ATP with an apparent kcat of approximately 19-22 min-1. Once the final binding state is achieved, binding and ATP hydrolysis by this complex becomes pH independent. The weak binding of recA protein to duplex DNA reported in previous studies does not, therefore, reflect an intrinsically unfavorable binding equilibrium. Instead, this apparent weak binding reflects a slow step in the association pathway. The rate-limiting step in this process involves the initiation rather than the propagation of DNA binding and unwinding. This step exhibits no dependence on recA protein concentration at pH 7.5. Extension or propagation of the recA filament is fast relative to the overall process. Initiation of binding is pH dependent and represents a prominent kinetic barrier at pH 7.5. ATP hydrolysis occurs only after the duplex DNA is unwound. The binding density of recA protein on double-stranded DNA is approximately one monomer/4 base pairs. A model for this process is presented. These results provide an explanation for several paradoxical observations about recA protein-promoted DNA strand exchange. In particular, they demonstrate that there is no thermodynamic requirement for dissociation of recA protein from the heteroduplex DNA product of strand exchange.

Adenosine Triphosphate↗
PubMed1987

recA protein binding to the heteroduplex product of DNA strand exchange.

Following DNA strand exchange, recA protein remains associated with the heteroduplex DNA product of the reaction. This association exists without significant changes (as measured by nuclease protection, topological state of the heteroduplex DNA, and rates of ATP hydrolysis) for at least 30 min after strand exchange is complete. The heteroduplex DNA is unwound as a result of this association to an extent at least as great as the unwinding observed when recA protein is bound to duplex DNA at pH 6.35. The extensive unwinding, in combination with the rates of ATP hydrolysis reported elsewhere, provide evidence that recA protein binding is contiguous throughout the heteroduplex DNA. This binding is disrupted upon challenge with heterologous single-stranded DNA, with rapid migration of recA protein to the challenging DNA. These results are discussed in relation to the mechanism of recA protein-promoted branch migration.

Adenosine Triphosphate↗
PubMed1986

Specific contacts between the FLP protein of the yeast 2-micron plasmid and its recombination site.

Contact points between the FLP protein of the yeast 2-micron plasmid and its recombination site have been defined. Important features of the region previously defined as the minimal recombination site in vitro include a pair of 13-base pair inverted repeats separated by an 8-base pair spacer. The two FLP protein-binding sites within this region are 12 base pairs in length. In each case they include the internal 11 base pairs of one of the 13-base pair repeats, as well as the adjacent base pair within the spacer. The internal 6 base pairs within the spacer are not involved in binding or recognition by FLP protein. When the size of the spacer is increased or decreased by one base pair, the distance between the cleavage points is also increased or decreased correspondingly by one base pair. Points of cleavage are unaffected by changes in the spacer sequence. Specific contact points involving purine residues, identified by methylation protection and recombination interference experiments, are located in both the major and minor grooves of the DNA. Additional contact points between FLP protein and phosphate groups in the phosphate-deoxyribose backbone are clustered near the cleavage sites.

Base Composition↗
PubMed1986

Exchange of recA protein between adjacent recA protein-single-stranded DNA complexes.

We have examined the exchange of recA protein between stable complexes formed with single-stranded DNA (ssDNA) and (a) other complexes and (b) a pool of free recA protein. We have also examined the relationship of ATP hydrolysis to these exchange reactions. Exchange was observed between two different recA X ssDNA complexes in the presence of ATP. Complete equilibration between two sets of complexes occurred with a t1/2 of 3-7 min under a set of conditions previously found to be optimal for recA protein-promoted DNA strand exchange. Approximately 200 ATPs were hydrolyzed for every detected migration of a recA monomer from one complex to another. This exchange occurred primarily between adjacent complexes, however. Little or no exchange was observed between recA X ssDNA complexes and the free recA protein pool, even after several hundred molecules of ATP had been hydrolyzed for every recA monomer present. ATP hydrolysis is not coupled to complete dissociation or association of recA protein from or with recA X ssDNA complexes under these conditions.

Adenosine Triphosphate↗
PubMed1986

Directionality in FLP protein-promoted site-specific recombination is mediated by DNA-DNA pairing.

The 2 mu plasmid of the yeast Saccharomyces cerevisiae encodes a site-specific recombination system consisting of plasmid-encoded FLP protein and two recombination sites on the plasmid. The recombination site possesses a specific orientation, which is determined by an asymmetric 8-base pair spacer sequence separating two 13-base pair inverted repeats. The outcome or directionality of site-specific recombination is defined by the alignment of two sites in the same orientation during the reaction. Sites containing point mutations or 1-base pair insertions or deletions within the spacer generally undergo recombination with unaltered sites at reduced levels. In contrast, recombination between the two identical mutant sites (where homology is restored) proceeds efficiently in all cases. Sites containing spacer sequences of 10 base pairs or more are nonfunctional under all conditions. A recombination site in which 5 base pairs are changed to yield an entirely symmetrical spacer sequence again recombines efficiently, but only with an identical site. This reaction, in addition, produces a variety of new products which can only result from random alignment of the two sites undergoing recombination, i.e. the reaction no longer exhibits directionality. These and other results demonstrate that both the efficiency and directionality of site-specific recombination is dependent upon homology between spacer sequences of the two recombining sites. This further implies that critical DNA-DNA interactions between the spacer region of the two sites involved in the reaction occur at some stage during site-specific recombination in this system. The specific spacer sequence itself appears to be unimportant as long as homology is maintained; thus, these sequences are probably not involved in recognition by FLP protein.

DNA↗
PubMed1986

Continuous association of Escherichia coli single-stranded DNA binding protein with stable complexes of recA protein and single-stranded DNA.

The single-stranded DNA binding protein of Escherichia coli (SSB) stimulates recA protein promoted DNA strand exchange reactions by promoting and stabilizing the interaction between recA protein and single-stranded DNA (ssDNA). Utilizing the intrinsic tryptophan fluorescence of SSB, an ATP-dependent interaction has been detected between SSB and recA-ssDNA complexes. This interaction is continuous for periods exceeding 1 h under conditions that are optimal for DNA strand exchange. Our data suggest that this interaction does not involve significant displacement of recA protein in the complex by SSB when ATP is present. The properties of this interaction are consistent with the properties of SSB-stabilized recA-ssDNA complexes determined by other methods. The data are incompatible with models in which SSB is displaced after functioning transiently in the formation of recA-ssDNA complexes. A continuous association of SSB with recA-ssDNA complexes may therefore be an important feature of the mechanism by which SSB stimulates recA protein promoted reactions.

Adenosine Diphosphate↗
PubMed1985

Light scattering studies of the recA protein of Escherichia coli: relationship between free recA filaments and the recA X ssDNA complex.

Light scattering has been used to monitor and distinguish between two types of aggregation reactions observed with the recA protein of Escherichia coli. These are (1) the cooperative binding of recA protein to ssDNA in a pathway leading to DNA strand exchange and (2) the formation of free filaments by recA protein in the absence of DNA. Free filament formation requires Mg2+, is very sensitive to ionic strength, and occurs in the absence of single-stranded DNA and RNA. Turbidity measurements indicate that free recA filaments exhibit properties consistent with rigid rods which are 1 micron or more in length. A kinetically distinct nucleation step in free filament formation is observed under some conditions and becomes rate limiting at high pH. Ninety-degree light scattering was employed to measure binding of recA protein to ssDNA under conditions that either favor or block free filament formation. recA protein saturates ssDNA at a stoichiometric ratio of approximately four nucleotide residues per recA monomer. When free filament formation is blocked by various means, the apparent dissociation constant of the recA X ssDNA complex is approximately 10 nM. Under conditions in which free recA filaments form readily, however, the apparent dissociation constant increases to approximately 1 microM. This dramatic decrease in the observed affinity of recA protein for ssDNA under conditions that permit free filament formation does not reflect a change in the intrinsic affinity of recA protein for ssDNA. Instead, it provides evidence that free filament formation and ssDNA binding by recA protein are competing reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Single-Stranded↗
PubMed1985

The FLP recombinase of the yeast 2-micron plasmid: characterization of its recombination site.

The minimal size of the recombination site required for efficient FLP recombinase-catalyzed recombination in vitro is no more than 28 base pairs, which includes parts of two 13-base-pair inverted repeats and all of an 8-base-pair spacer. The FLP recombinase cleaves the DNA at the boundaries of the spacer, becomes covalently linked to the spacer DNA via a 3' phosphate, and leaves a free 5' hydroxyl at the other end of the 8-base-pair spacer. The efficiency of recombination is reduced if the size of the spacer in a recombinant site is increased or decreased by 1 base pair, while the spacer in the second site is unaltered. Recombination between two sites with identical 1-base-pair additions or deletions in the spacer, however, is relatively unaffected. This result suggests that pairing of sequences in the spacer region is important in FLP-promoted recombination events. The sequence asymmetry utilized by the recombinase to determine the orientation of the site is located uniquely within the spacer region.

Base Sequence↗
PubMed1984

Unidirectional branch migration promoted by nucleoprotein filaments of RecA protein and DNA.

Described above are the initial results from a series of experiments designed to more carefully define the mechanism of RecA protein-promoted DNA strand exchange, and in particular, the branch migration phase of this reaction. We have also presented two general models for RecA protein-promoted branch migration. Although the data required to demonstrate a particular mechanism are presently unavailable, we believe these models will provide a useful framework to suggest future experiments. The models also suggest possible explanations for observations that are presently unexplained. In particular, it is possible to explain the observed inefficiency of ATP hydrolysis in this system without invoking the notion that much of the ATP hydrolysis observed is simply uncoupled. If the utilization of extensive filaments of RecA protein to carry out branch migration is inherently inefficient, why might such a mechanism be employed by the cell? The answer to this question may lie in the role of RecA protein-promoted branch migration in post-replication repair (West et al. 1981c). It might be expected that the branch points in a RecA protein-promoted branch migration reaction are susceptible to nuclease degradation. Such degradation would halt branch migration and prevent postreplication repair. One of the roles of the RecA nucleoprotein filament may be to protect the branch point from nuclease degradation and ensure formation of an extensive region of heteroduplex DNA. The apparent energetic cost of this reaction may be a relatively small price to pay to ensure the repair of a potentially lethal DNA lesion.

DNA, Bacterial↗
PubMed1983

On the role of single-stranded DNA binding protein in recA protein-promoted DNA strand exchange.

RecA protein-promoted DNA strand exchange is greatly stimulated by the single-stranded DNA binding protein (SSB) of Escherichia coli. Stimulation is not a consequence of the binding of SSB to excess single-stranded DNA. It results instead from stabilization of recA protein-single-stranded DNA complexes formed in the presence of ATP and SSB. In the presence of SSB, recA protein does not measurably dissociate from these complexes for up to 90 min. However, in its absence, recA protein moves rapidly between two populations of single-stranded DNA, and complete equilibration occurs with t 1/2 of 17 s. Rapid transfer of recA protein to single-stranded DNA occurs during all stages of DNA strand exchange and does not require ATP. The transfer involves an equilibrium between free and bound recA protein rather than a direct redistribution between single-stranded DNA molecules. Thus, SSB prevents dissociation of recA protein from single-stranded DNA, rendering the binding of the recA protein to single-stranded DNA irreversible. Under these conditions, the pairing phase of the strand exchange reaction is accelerated to the point that it is no longer rate-limiting. These results can explain the relative inefficiency of DNA strand exchange in the absence of SSB.

Bacterial Proteins↗
PubMed1983

ADP-mediated dissociation of stable complexes of recA protein and single-stranded DNA.

The complete exchange of strands between circular single-stranded and full length linear duplex DNAs promoted by the recA protein of Escherichia coli is dependent upon the hydrolysis of ATP and is strongly stimulated by the single-stranded DNA binding protein (SSB). In the presence of SSB, stable complexes of recA protein and single-stranded DNA are formed as an early step in the reaction. These complexes dissociate when the ADP/ATP ratio approaches a value of 0.6-1.5, depending upon reaction conditions. Thus, ATP hydrolysis never proceeds to completion but stops when 40-60% of the input ATP has undergone hydrolysis. recA protein can participate in a second round of strand exchange upon regeneration of the ATP. While 100-200 mol of ATP are hydrolyzed/mol of heteroduplex base pair formed under standard reaction conditions in the presence of SSB, this value is reduced to 16 at levels of ADP lower than that required to dissociate the complexes. ATP hydrolysis appears to be completely irreversible since efforts to detect exchange reactions using 18O probes have been unsuccessful.

Adenosine Diphosphate↗
PubMed1983

The FLP protein of the yeast 2-microns plasmid: expression of a eukaryotic genetic recombination system in Escherichia coli.

The FLP gene of the yeast 2-microns plasmid is involved in a site-specific recombination event that results in the inversion of a set of sequences within the plasmid. This gene has been cloned and expressed in Escherichia coli. Expression of the FLP gene results in efficient recombination within the bacterial cell, which is specific for plasmids containing at least one 2-microns plasmid recombination site. This work demonstrates that (i) FLP protein is actively involved in 2-microns plasmid recombination; (ii) no other factors specific to yeast are required for the reaction; (iii) FLP protein acts efficiently in trans; (iv) FLP protein will promote site-specific insertion and deletion reactions in addition to the inversion reaction; and (v) FLP-promoted recombination is not dependent upon any DNA structural features unique to yeast chromatin.

Base Sequence↗
PubMed1982

recA protein-promoted DNA strand exchange. Stable complexes of recA protein and single-stranded DNA formed in the presence of ATP and single-stranded DNA binding protein.

The recA protein of Escherichia coli promotes the complete exchange of strands between full length linear duplex and single-stranded circular DNA molecules. An early step in this reaction consists of the binding of recA protein to single-stranded DNA. In the presence of ATP and the single-stranded DNA binding protein, recA protein and single-stranded DNA interact to form a complex whose stability depends upon the single-stranded DNA binding protein. Duplex DNA is not required for complex formation. Subsequent steps occur within this complex which contains up to 1 recA protein monomer per 2 nucleotides of single-stranded DNA. Although several hundred ATPs are hydrolyzed per recA protein monomer, recA protein is not released from the complex at any step during or subsequent to strand exchange. The complex is kinetically competent with respect to both its rate of formation and its rate of reaction with homologous duplex DNA. It is therefore a significant intermediate in the overall reaction pathway. These results have served as the basis for an expanded model for recA protein-promoted strand exchange.

Adenosine Triphosphate↗