Mutant RecA proteins which form hexamer-sized oligomers 1 1Edited by M. F. Moody

Logan, Karen M.; Skiba, Mark C.; Eldin, Sherif; Knight, Kendall L.

doi:10.1006/jmbi.1996.0751

Cited by 18 publications

(22 citation statements)

References 34 publications

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“…Determination of the Size of Mutant RecA Protein Oligomers-The oligomeric distribution of wild type and mutant RecA proteins was determined using gel filtration chromatography as described previously (19).…”

Section: Materials-labeledmentioning

confidence: 99%

Allosteric Regulation of RecA Protein Function Is Mediated by Gln194

Kelley

Knight

1997

Journal of Biological Chemistry

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Binding of ATP to the RecA protein induces a high affinity DNA binding required for activation of enzyme function. Screens for in vivo recombination and repressor cleavage activities show Gln 194 to be intolerant of all substitutions. Analyses of three mutant proteins (Q194N, Q194E, and Q194A) show that although basal enzyme function is maintained, each protein no longer displays an ATP-induced increase in DNA binding affinity. High salt activation of RecA function is also disrupted by these mutations. In contrast, ATP-induced changes in the oligomeric structure of RecA are maintained in the mutant proteins. These results demonstrate that Gln 194 is a critical "allosteric switch" for ATP-induced activation of RecA function but is not the exclusive mediator of ATP-induced changes in RecA.The Escherichia coli RecA protein is a multifunctional enzyme that plays a central role in the processes of recombinational DNA repair, homologous genetic recombination, and the cellular SOS response to DNA damage (1-3). Each of these activities exhibits a common initial or activating step, formation of a RecA-ATP-ssDNA 1 nucleoprotein filament (4 -6). The binding of RecA to ssDNA is regulated in a classic allosteric fashion, whereby the binding of ATP induces a high affinity DNA binding state of the protein (7,8). In the presence of ADP, or in the absence of cofactor, RecA exhibits a low affinity (Ͼ20 M) for ssDNA (7).The crystal structure of the helical RecA protein filament has been solved in both the absence and presence of ADP (9, 10) and displays a helical pitch (82.7 Å) which is intermediate between the inactive nucleotide-free form (Ϸ70 Å) and the active ATPbound form (Ϸ95 Å) as determined by electron microscopy (reviewed in Ref. 11). Despite this, the ATP binding site shows a remarkable conservation of structure compared with several other nucleotide-binding proteins (e.g. p21 ras , EF-Tu, and adenylate kinase), and Story and Steitz (10) were able to model specific determinants of both ATP binding and hydrolysis in the RecA structure. In addition, the structure provided valuable insight into a possible allosteric mechanism for ATP-induced high affinity binding to ssDNA. The carboxyamide side chain of Gln 194 extends into the ATP binding site and would be in very close proximity to the ␥-phosphate of bound ATP (Fig. 1). Gln 194 immediately precedes one of two disordered loops in the structure (L2, residues 195-209) that were proposed to be part of the DNA binding sites (9). Recent work has, in fact, provided strong evidence that L2 comprises all or a large part of the ssDNA binding site within . Story and Steitz (10) proposed that upon ATP binding Gln 194 interacts with the nucleotide ␥-phosphate thereby causing L2 to assume a conformation with high affinity for ssDNA. Upon ATP hydrolysis this interaction would be lost, returning L2 to a low affinity DNA binding conformation.In is an important "on-off" switch required for the general activation of RecA function. EXPERIMENTAL PROCEDURES Materials-LabeledNTPs and dNTPs wer...

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Section: Materials-labeledmentioning

confidence: 99%

Allosteric Regulation of RecA Protein Function Is Mediated by Gln194

Kelley

Knight

1997

Journal of Biological Chemistry

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“…In vivo, two types of RecA-GFP structures were identified: those that are on the DNA (about 50% of the structures) and those that are not (the remaining 50%). The latter are presumably storage structures (16,25,29). It has been shown that recA(R28A) is proficient for recombination and DNA repair in vivo but does not make storage structures in vitro (7).…”

mentioning

confidence: 99%

UvrD Limits the Number and Intensities of RecA-Green Fluorescent Protein Structures in Escherichia coli K-12

Centore

Sandler

2007

J Bacteriol

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RecA is important for recombination, DNA repair, and SOS induction. In Escherichia coli, RecBCD, RecFOR, and RecJQ prepare DNA substrates onto which RecA binds. UvrD is a 3-to-5 helicase that participates in methyl-directed mismatch repair and nucleotide excision repair. uvrD deletion mutants are sensitive to UV irradiation, hypermutable, and hyper-rec. In vitro, UvrD can dissociate RecA from singlestranded DNA. Other experiments suggest that UvrD removes RecA from DNA where it promotes unproductive reactions. To test if UvrD limits the number and/or the size of RecA-DNA structures in vivo, an uvrD mutation was combined with recA-gfp. This recA allele allows the number of RecA structures and the amount of RecA at these structures to be assayed in living cells. uvrD mutants show a threefold increase in the number of RecA-GFP foci, and these foci are, on average, nearly twofold higher in relative intensity. The increased number of RecA-green fluorescent protein foci in the uvrD mutant is dependent on recF, recO, recR, recJ, and recQ. The increase in average relative intensity is dependent on recO and recQ. These data support an in vivo role for UvrD in removing RecA from the DNA.

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“…Light scattering studies have found alternative species that have been formed in the presence of Mg 2+ ions [17,23]. Additionally, FPLC measurements detect alternative states of aggregation that are not explained by results from EM [9]. Without corroboration from an imaging technique, the observation of these aggregates has been set aside in many reports on recA, these results take on a renewed importance in light of the true physiological result obtained here by AFM.…”

Section: Discussionmentioning

confidence: 70%

“…Electron microscopy (EM) [7][8][9][10][11][12], scanning tunnelling microscopy [13,14], light scattering (LS) [15] and small angle neutron scattering (SANS) [16] have been utilised to show the gross morphology and aggregation of recA monomers with each other, and with DNA. A variety of structures have been observed: hexamers, short rods, bundles of rods…”

Section: Introductionmentioning

confidence: 99%

“…Further examination using quick freeze/deep etch EM found recA to collapse into bundles in the presence of spermidine [19]. FPLC found recA also to exist in distinct aggregation states [9]. The presence, and proportions, of the various aggregates were also shown to be dependent on the concentration of recA and the incubation temperature; the fibrils at the highest concentrations were typically up to 200 nm in length [21].…”

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confidence: 98%

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Novel Polymorphism of RecA Fibrils Revealed by Atomic Force Microscopy

Sattin

Goh

2006

J Biol Phys

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Abstract. RecA fibrils in physiological conditions have been successfully imaged using Tapping Mode atomic force microscopy. This represents the first time images of recA have been obtained without drying, freezing and/or exposure to high vacuum conditions. While previously observed structuresthe monomer, the hexamer, the short rod -were seen, a new type of fibril was also observed. This protofibril is narrower in diameter than the standard fibril, and occurs in three distinct morphologies: aperiodic, 100-nm periodic, and 150-nm periodic. In addition, much longer rods were observed, and appear curved and even circular. Key words:RecA, recombination, assembly, AFMThe protein recA and its associated analogues are critically involved in a number of biological processes, centering around strand exchange activity: in the model organism E. coli, it serves as an essential enzyme in both the SOS mutagenesis pathway and in homologous recombination [1,2]. There has been renewed interest in this prokaryotic protein due to the discovery of many analogues in a wide diversity of systems, e.g., Rad51 in eukaryotes [3], and pk-REC in archaea [4]. These analogues were shown to perform much the same function in these organisms as recA does in E. coli. In addition, recA has a strong sequence homology to typical motor proteins, such as the F 1 ATPase, and dmc1 [1]. High concentrations of aggregated recA have been found in the bacterial spliceosome -a postulated cellular centre for genetic repair and recombination [5]. Hence, the study of the recA protein has assumed an increased importance, and a multitude of different techniques have been employed in order to elucidate its structure and function.The atomic structure of the E. coli recA/ADP complex has been resolved by X-ray crystallography to 2.3Å resolution, showing the atomic monomer structure, and proposed dimensions for the hexamer structure [6]. Electron microscopy (EM) [7][8][9][10][11][12], scanning tunnelling microscopy [13,14], light scattering (LS) [15] and small angle neutron scattering (SANS) [16] have been utilised to show the gross morphology and aggregation of recA monomers with each other, and with DNA. A variety of structures have been observed: hexamers, short rods, bundles of rods

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Mutant RecA proteins which form hexamer-sized oligomers 1 1Edited by M. F. Moody

Cited by 18 publications

References 34 publications

Allosteric Regulation of RecA Protein Function Is Mediated by Gln194

Allosteric Regulation of RecA Protein Function Is Mediated by Gln194

UvrD Limits the Number and Intensities of RecA-Green Fluorescent Protein Structures in Escherichia coli K-12

Novel Polymorphism of RecA Fibrils Revealed by Atomic Force Microscopy

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