Isolation, characterization, and magnesium-induced self-association kinetics of discrete aggregates of RecA protein from Escherichia coli

Budzynski, Donna M.; Gao, Xiang; Benight, Albert S.

doi:10.1002/(sici)1097-0282(199604)38:4<471::aid-bip4>3.0.co;2-q

Cited by 7 publications

(5 citation statements)

References 41 publications

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“…Many studies have shown that the oligomeric state of free RecA protein is exquisitely sensitive to a variety of solution conditions including protein concentration, ionic strength, and temperature and that at the protein concentrations typically used in DNA strand exchange assays, RecA exists as a complex mix of various oligomeric forms (9)(10)(11)(12)(27)(28)(29)(30). The fact that the RecA-FD protein functions both in ViVo and in Vitro demonstrates that dimeric RecA can serve as the nucleating unit for filament assembly onto ssDNA.…”

Section: Discussionmentioning

confidence: 99%

RecA Dimers Serve as a Functional Unit for Assembly of Active Nucleoprotein Filaments

et al. 2006

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All RecA-like recombinase enzymes catalyze DNA strand exchange as elongated filaments on DNA. Despite numerous biochemical and structural studies of RecA and the related Rad51 and RadA proteins, the unit oligomer(s) responsible for nucleoprotein filament assembly and coordinated filament activity remains undefined. We have created a RecA fused dimer protein and show that it maintains in vivo DNA repair and LexA co-protease activities, as well as in vitro ATPase and DNA strand exchange activities. Our results support the idea that dimeric RecA is an important functional unit both for assembly of nucleoprotein filaments as well as their coordinated activity during the catalysis of homologous recombination.The bacterial RecA protein is the founding member of a family of enzymes that catalyzes recombination between homologous DNA substrates, a family which includes the phage T4 UvsX, yeast Rad51, archael RadA and human Rad51 proteins (1,2). To perform this function these recombinase enzymes form a helical filament that assembles cooperatively on a singlestranded region of DNA resulting in the formation of an active nucleoprotein complex that subsequently interacts with a dsDNA substrate to catalyze a search for homology and DNA strand exchange (1,3,4). Early biochemical studies of RecA revealed that in the absence of substrate DNA the protein exists in solution as a complex heterogeneous mix of various oligomers (5-8) and later work showed that this mix included monomers, ring-shaped hexamers/heptamers, short filaments ranging in length from 0.03 to 0.15 μm as well as bundles of filaments (9-12).The RecA nucleoprotein filament assembles in an ATP-dependent, highly cooperative manner (13-16). Kinetic analysis of nucleoprotein filament assembly has shown that RecA polymerization onto ssDNA can be divided into two major steps, i. nucleation, which is rate limiting, and ii. filament extension (17). However, the oligomeric heterogeneity of RecA in these studies precluded identification of a specific RecA oligomer, e.g. monomers or dimers, as being an obligatory component of the assembly process, or whether various larger RecA oligomers, e.g. trimers or hexamers, could participate in filament assembly. It was also found that RecA oligomers formed in the absence of ATP or ssDNA are not directly interconvertible tel. -(508) EXPERIMENTAL PROCEDURES Expression ConstructsThe gene encoding wild type recA was expressed using a previously described construct, pTRecA420, in which recA is regulated by the tac promoter (20,21). The gene encoding the fused RecA dimer (recA-FD) was constructed as follows. Two point mutations, Lys6Ala and Arg28Ala, were introduced into the wild type recA gene carried in plasmid pTRecA420 using the QuikChange protocol (Stratagene). This mutant recA gene was PCR amplified using a top strand primer that overlapped the NcoI restriction site (underlined) at the fMet initiation codon (5′-GG AGT GAT GCC ATG GCT ATC GAC G -3′) and a bottom strand primer that adds a five-residue linker (Gly 3 Ser 2 )...

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Section: Discussionmentioning

confidence: 99%

RecA Dimers Serve as a Functional Unit for Assembly of Active Nucleoprotein Filaments

et al. 2006

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“…RecA exists in numerous forms consisting of oligomers, helical filaments or filamentous bundles that are dependent on the solution conditions, concentration of RecA, and the presence of cofactors including nucleotides and/or DNA (Roca and Cox, 1997;Brenner et al, 1988Brenner et al, , 1990Budzynski et al, 1996;Egelman, 1993). The binding of nucleotides by RecA has been characterized by at least two different DNA binding states.…”

Section: Introductionmentioning

confidence: 99%

Investigating Structural Changes Induced By Nucleotide Binding to RecA Using Difference FTIR

Butler

Hanchett

Rafailov

et al. 2002

Biophysical Journal

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Nucleotide binding to RecA results in either the high-DNA affinity form (Adenosine 5'-triphosphate (ATP)-bound) or the more inactive protein conformation associated with a lower affinity for DNA (Adenosine 5'-diphosphate (ADP)-bound). Many of the key structural differences between the RecA-ATP and RecA-ADP bound forms have yet to be elucidated. We have used caged-nucleotides and difference FTIR in efforts to obtain a comprehensive understanding of the molecular changes induced by nucleotide binding to RecA. The photochemical release of nucleotides (ADP and ATP) from biologically inactive precursors was used to initiate nucleotide binding to RecA. Here we present ATP hydrolysis assays and fluorescence studies suggesting that the caged nucleotides do not interact with RecA before photochemical release. Furthermore, we now compare difference spectra obtained in H2O and D2O as our first attempt at identifying the origin of the vibrations influenced by nucleotide binding. The infrared data suggest that unique alpha-helical, beta structures, and side chain rearrangements are associated with the high- and low-DNA affinity forms of RecA. Difference spectra obtained over time isolate contributions arising from perturbations in the nucleotide phosphates and have provided further information about the protein structural changes involved in nucleotide binding and the allosteric regulation of RecA.

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“…However, the state of aggregation is sensitive to environmental conditions, − and the nature of the fiber inside a gel is not known. Furthermore, at least oligomeric states of the fiber are rather dynamic at room temperature, so it is uncertain whether the RecA-only fiber will remain intact during migration through a gel.…”

Section: Resultsmentioning

confidence: 99%

Gel-Shift Assays: Migrative Dissociation of a RecA−Oligonucleotide Complex during Electrophoresis in Hydroxyethylated Agarose Gels

2001

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We investigate the dynamics of a protein-DNA complex under gel-shift assay conditions, in particular how the degree of gel confinement influences the complex stability during the electrophoretic analysis. The fiberlike complex between RecA proteins and a single-stranded oligonucleotide has been studied in hydroxyethylated agarose gels with the average pore radius being between 1 and 5 times larger than the radius of the rodshaped complex. The confining effect of the gel matrix slows down the dissociation of the complex, but in addition, migrative interactions (collisions) with the gel network during the electrophoresis perturbs the complex in two ways. At low gel concentrations, the protein-DNA complex is dismantled into free RecA and oligonucleotide with a half-time of the complex that decreases with increasing field strength in accordance with a migrative mechanism. At a given field strength, the half-time increases with increasing gel concentration despite more frequent interactions, probably because of a counteracting stabilizing cage effect from the gel. At high gel concentrations, a second type of perturbation is reflected in an increasing complex velocity over time, probably caused by trains of end-to-end attached fibers being broken up by interactions with the tight gels.

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Isolation, characterization, and magnesium-induced self-association kinetics of discrete aggregates of RecA protein from Escherichia coli

Cited by 7 publications

References 41 publications

RecA Dimers Serve as a Functional Unit for Assembly of Active Nucleoprotein Filaments

RecA Dimers Serve as a Functional Unit for Assembly of Active Nucleoprotein Filaments

Investigating Structural Changes Induced By Nucleotide Binding to RecA Using Difference FTIR

Gel-Shift Assays: Migrative Dissociation of a RecA−Oligonucleotide Complex during Electrophoresis in Hydroxyethylated Agarose Gels

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