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

Abstract

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...

ADP stabilizes the human Rad51‐single stranded DNA complex and promotes its DNA annealing activity

“…2B), and was 1.6‐fold larger than that of the complex without nucleotide. The effect of ADP on the HsRad51‐poly(dɛA) complex differs from that on the RecA‐ or XRad51‐poly(dɛA) complexes: ADP strongly decreases the fluorescence from the RecA‐poly(dɛA) complex (Ellouze et al . 1999), and does not significantly change that of the XRad51‐poly(dɛA) complex (Maeshima et al .…”

The SOS system: A complex and tightly regulated response to DNA damage

“…In vitro , other nucleotide triphosphates are competitive inhibitors of dATP‐promoted RecA coprotease activity (Weinstock and McEntee, ; Weinstock et al, ). It was suggested that the chemical nature of the nucleobase is important for the stability of the RecA‐ssDNA complex: adenine promotes strong RecA‐DNA binding, while other bases promote only weak binding, leading to enhanced dissociation of the filament (Ellouze et al, ). Of the two possible RecA filament states, only one explains the interaction between LexA and RecA that promotes LexA cleavage (VanLoock et al, ), and the efficient formation of RecA filaments in the active conformation is necessary for SOS induction (Gruenig et al, ).…”

“…Little in vivo information is available about the possible role of the cellular dNTPs in RecA activation, although a number of studies have addressed the role of alternative cofactors using in vitro experiments (Phizicky and Roberts, 1981;Weinstock and McEntee, 1981;Menetski et al, 1988;Wang et al, 1988a;Ellouze et al, 1999;Wigle et al, 2006). Various (d)NTP species show different efficiencies in promoting RecA coprotease activity in vitro (Phizicky and Roberts, 1981;Weinstock et al, 1981a).…”

RecA‐mediated SOS induction requires an extended filament conformation but no ATP hydrolysis

“…Other nucleotides are available in vivo , and in vitro experiments show that RecA can bind several of them and retain at least some key functions (McEntee et al ., 1981; Weinstock et al ., 1981a,b,c; Menge and Bryant, 1988; 1992a,b). The nucleotide dATP substitutes best for ATP in vitro , and several RecA functions are enhanced when dATP replaces ATP in reaction mixtures (Menetski and Kowalczykowski, 1989; Shan et al ., 1997; Ellouze et al ., 1999; Robu et al ., 2001).…”