Stable DNA heteroduplex formation catalyzed by the Escherichia coli RecA protein in the absence of ATP hydrolysis.

Abstract

A question remaining to be answered about RecA protein function concerns the role of ATP hydrolysis during the DNA-strand-exchange reaction. In this paper we describe the formation ofjoint molecules in the absence of ATP hydrolysis, using adenosine 5'-[r-thio]triphosphate (ATP[yS]) as nucleotide cofactor. Upon the addition of double-stranded DNA, the ATP[ySJ-RecA protein-single-stranded DNA presynaptic complexes can form homologously paired molecules that are stable after deproteinization. Formation of these joint molecules requires both homology and a free homologous end, suggesting that they are plectonemic in nature. This reaction is very sensitive to magnesium ion concentration, with a maximum rate and extent observed at 4-5 mM magnesium acetate. Under these conditions, the average length of heteroduplex DNA within the joint molecules is 2.4-3.4 kilobase pairs. Thus, RecA protein can form extensive regions of heteroduplex DNA in the presence of ATP [yS], suggesting that homologous pairing and the exchange of the DNA molecules can occur without ATP hydrolysis. A model for the RecA protein-catalyzed DNA-strand-exchange reaction that incorporates these results and its relevance to the mechanisms ofeukaryotic recombinases are presented.The RecA protein-catalyzed DNA-strand-exchange reaction has been divided into at least three distinct phases (1, 2). In the first phase of this reaction, RecA protein binds to single-stranded DNA (ssDNA) in the presence of ATP (presynapsis). During the second phase, synapsis, the ssDNA and double-stranded DNA (dsDNA) (4)]. Since plectonemic, as well as paranemic, joint molecules can be formed in the presence of ATP, it was suggested that conversion of paranemic to plectonemic joint molecules requires ATP hydrolysis (4). In related studies, addition ofATP[yS] to an ongoing DNA-strand-exchange reaction was shown to immediately stop formation of heteroduplex DNA (3). The data were interpreted to suggest that ATP hydrolysis was also required for the formation of extensive regions of heteroduplex DNA (after joint molecules are formed). Thus, homologous pairing and the formation of protein-stabilized paranemic joint molecules are thought to occur in the absence of ATP hydrolysis, whereas the formation of plectonemic joint molecules and extensive regions of heteroduplex DNA requires hydrolysis of ATP. We now report the catalysis ofDNA strand exchange in the presence of ATP [yS]. Our data demonstrate that the joint molecules formed in the presence ofATP [yS] are stable in the absence of RecA protein and are, therefore, plectonemic in nature. The average length of heteroduplex DNA in these joint molecules can be as much as 3.4 kilobase pairs (kb) with no detectable hydrolysis of ATP [yS]. The relevance of these results to the role of ATP hydrolysis in the mechanism of the DNA strand exchange catalyzed by the Escherichia coli RecA protein and by other recombinases is discussed. MATERIALS AND METHODSReagents. All chemicals used were reagent grade and solutions were made in gla...

Domain structure and dynamics in the helical filaments formed by RecA and Rad51 on DNA

“…The smooth helical backbone appears relatively unchanged. Whereas ATP-␥-S, even though is is slowly hydrolyzed, has been shown in many structural and biochemical studies with the RecA protein to be a good analog for ATP (32,33), our observations indicate that ATP-␥-S and ATP induce two different filamentous states of the hRad51 protein on ssDNA. This finding is consistent with biochemical observations suggesting that ATP-␥-S and ATP behave differently with respect to the Rad51 and RecA proteins.…”

Base pair switching by interconversion of sugar puckers in DNA extended by proteins of RecA-family: A model for homology search in homologous genetic recombination

“…We propose that the DNA structure with the deoxyribose-base stacking has sufficient flexibility to allow the spontaneous conformational change required for base pair switching. This flexible property would be of advantage to the process of homologous pairing, because homologous pairing occurs very quickly without exogenous energy supply such as ATP hydrolysis (59)(60)(61). The base rotation through the conversion of sugar puckers might be driven by thermodynamic molecular motions and could progress easily by using the energy provided by external heat under physiological temperature.…”

“…Various biochemical studies indicated that homologous pairing and strand exchange can occur by using the nonhydrolyzable ATP analog, ATP␥S (60,61), or by a mutant RecA protein (RecA K72R) that exhibits Ͼ600-fold reduced level of an NTP hydrolysis (62,63). Active RecA filaments formed in the presence of both ATP and DNA exhibit an extended structure with helical pitch of Ϸ95 Å (19,(31)(32)(33), whereas in the presence of ADP or the absence of nucleotide cofactors, RecA protein formed collapsed filaments with a helical pitch of Ϸ64 Å (64-68).…”

Magnesium Ion-dependent Activation of the RecA Protein Involves the C Terminus

“…However, the initiation of DNA pairing in the presence of ATP␥S should occur in a manner similar to the more extended DNA strand exchange. As is the case for the DNA strand exchange reaction with ATP, the optimal conditions for RecA protein-promoted formation of joint molecule intermediates in the presence of ATP␥S include magnesium ion concentrations in significant excess relative to the ATP␥S that is present (30). We examined the effect of DNA overhangs on both wild-type RecA and RecA⌬C17-promoted DNA strand exchange reactions.…”