Evidence for nucleotide-mediated changes in the domain structure of the RecA protein of Escherichia coli

Kobayashi, N.; Knight, Kendall L.; McEntee, Kevin

doi:10.1021/bi00395a033

Cited by 42 publications

(16 citation statements)

References 35 publications

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“…Partial proteolysis has been a useful method for determining conformational transitions associated with nucleotide-binding proteins, including RecA (44). hRAD51 was preincubated with ADP, ATP, or ATP␥S and subsequently exposed to a limiting amount of either endoprotease Lys-C or trypsin.…”

Section: Adenosine Nucleotides Induce Conformational Transitions In Hmentioning

confidence: 99%

Biochemical Characterization of the Human RAD51 Protein

Tombline

Heinen

Shim

et al. 2002

Journal of Biological Chemistry

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Adenosine nucleotides affect the ability of RecA⅐single-stranded DNA (ssDNA) nucleoprotein filaments to cooperatively assume and maintain an extended structure that facilitates DNA pairing during recombination. Here we have determined that ADP and ATP/ATP␥S affect the DNA binding and aggregation properties of the human RecA homolog human RAD51 protein (hRAD51). These studies have revealed significant differences between hRAD51 and RecA. In the presence of ATP␥S, RecA forms a stable complex with ssDNA, while the hRAD51 ssDNA complex is destabilized. Conversely, in the presence of ADP and ATP, the RecA ssDNA complex is unstable, while the hRAD51 ssDNA complex is stabilized. We identified two hRAD51⅐ssDNA binding forms by gel shift analysis, which were distinct from a well defined RecA⅐ssDNA binding form. The available evidence suggests that a low molecular weight hRAD51⅐ssDNA binding form (hRAD51⅐ssDNA low ) correlates with active ADP and ATP processing. A high molecular weight hRAD51⅐ ssDNA aggregate (hRAD51⅐ssDNA high ) appears to correlate with a form that fails to process ADP and ATP. Our data are consistent with the notion that hRAD51 is unable to appropriately coordinate ssDNA binding with adenosine nucleotide processing. These observations suggest that other factors may assist hRAD51 in order to mirror RecA recombinational function.

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Section: Adenosine Nucleotides Induce Conformational Transitions In Hmentioning

confidence: 99%

Biochemical Characterization of the Human RAD51 Protein

Tombline

Heinen

Shim

et al. 2002

Journal of Biological Chemistry

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“…A few investigations suggested that the disordered L1 loop of RecA is exposed upon ATP binding (Kobayashi et al, 1987;. The proximity of the L1 loop to the mutation site of RecA423 supports a role for position 160 in a conformational change.…”

Section: In Vitro Properties Of Reca423 Proteinmentioning

confidence: 99%

Characterization of a Mutant RecA Protein that Facilitates Homologous Genetic Recombination but not Recombinational DNA Repair: RecA423

Ishimori¹,

Sommer

Bailone

et al. 1996

Journal of Molecular Biology

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“…The activation of RecA by ATP can be explained by an allosteric mechanism. The activating nucleotides, ATP and ATP[S], affect the conformation of RecA protein [18–20], and modify the structure of the RecA filament [11,21–23] and the DNA binding mode [11,24–26]. In the presence of ATP or its analog, the RecA filament is elongated with an increase in the size of the helical pitch and a decrease in the diameter, DNA binding affinity of RecA is increased, and the nucleobases of DNA bound to RecA become less mobile and are oriented in one direction.…”

mentioning

confidence: 99%

Difference between active and inactive nucleotide cofactors in the effect on the DNA binding and the helical structure of RecA filament

Ellouze

Selmane

Kim

et al. 1999

European Journal of Biochemistry

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The RecA protein requires ATP or dATP for its coprotease and strand exchange activities. Other natural nucleotides, such as ADP, CTP, GTP, UTP and TTP, have little or no activation effect on RecA for these activities. We have investigated the activation mechanism, and the selectivity for ATP, by studying the effect of various nucleotides on the DNA binding and the helical structure of the RecA filament. The interaction with DNA was investigated via fluorescence measurements with a fluorescent DNA analog and fluorescein-labeled oligonucleotides, assisted by linear dichroism. Filament structure was investigated via small-angle neutron scattering. There is no simple correlation between filament elongation, DNA binding affinity of RecA, and DNA structure in the RecA complex. There may be multiple conformations of RecA. Both coprotease and strand exchange activities require formation of a rigid and well organized complex. The triphosphate nucleotides which do not activate RecA, destabilize the RecA±DNA complex, indicating that the chemical nature of the nucleotide nucleobase is very important for the stability of RecA±DNA complex. Higher stability of the RecA-DNA complex in the presence of adenosine 5 H -O-3-thiotriphosphate or guanosine 5 H -O-3-thiotriphosphate than ATP or GTP indicates that contact between the protein and the chemical group at the gamma position of the nucleotide also affects the stability of the RecA±DNA complex. This contact appears also important for the rigid organization of DNA because ADP strongly decreases the rigidity of the complex.Keywords: RecA protein; homologous recombination; nucleotide cofactor; DNA binding; RecA filament.RecA protein is a cardinal element of the DNA repair system in Escherichia coli [1±3]. The protein regulates the synthesis of DNA repair enzymes (SOS induction), catalyzes homologous recombination and is involved in mutagenesis. Purified RecA mimics these activities in vitro: RecA catalyzes strand exchange between two homologous DNA molecules [4,5], stimulates autocleavage of LexA repressor [6,7] and UmuD protein [8], and interacts with UmuD H protein [9]. For these activities, RecA requires ATP or dATP as a cofactor and interacts with DNA with very high cooperativity to form a filamentous complex, in which RecA subunits are organized in a helical manner around the DNA [10,11]. The activation of RecA occurs only with ATP or dATP. Other natural nucleotides, ADP, CTP, GTP or TTP do not activate RecA [5,12±14], even though they interact with [13,15] and are hydrolyzed by RecA [14,16].Hydrolysis of the nucleotide is required neither for strand exchange [17] nor coprotease activity [12,13]. Both these reactions can be promoted by a nonhydrolysable analog, adenosine 5 H -O-3-thiotriphosphate (ATP [S]). The activation of RecA by ATP can be explained by an allosteric mechanism. The activating nucleotides, ATP and ATP [S], affect the conformation of RecA protein [18±20], and modify the structure of the RecA filament [11,21±23] and the DNA binding mode [11,24±26]. In the pr...

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Evidence for nucleotide-mediated changes in the domain structure of the RecA protein of Escherichia coli

Cited by 42 publications

References 35 publications

Biochemical Characterization of the Human RAD51 Protein

Biochemical Characterization of the Human RAD51 Protein

Characterization of a Mutant RecA Protein that Facilitates Homologous Genetic Recombination but not Recombinational DNA Repair: RecA423

Difference between active and inactive nucleotide cofactors in the effect on the DNA binding and the helical structure of RecA filament

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