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...

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The DNA-dependent protein kinase (DNA-PK) is required for DNA double-strand break (DSB) repair and immunoglobulin gene rearrangement and may play a role in the regulation of transcription. The DNA-PK holoenzyme is composed of three polypeptide subunits: the DNA binding Ku70͞86 heterodimer and an Ϸ460-kDa catalytic subunit (DNA-PKcs). DNA-PK has been hypothesized to assemble at DNA DSBs and play structural as well as signal transduction roles in DSB repair. Recent advances in atomic force microscopy (AFM) have resulted in a technology capable of producing high resolution images of native protein and proteinnucleic acid complexes without staining or metal coating. The AFM provides a rapid and direct means of probing the protein-nucleic acid interactions responsible for DNA repair and genetic regulation. Here we have employed AFM as well as electron microscopy to visualize Ku and DNA-PK in association with DNA. A significant number of DNA molecules formed loops in the presence of Ku. DNA looping appeared to be sequence-independent and unaffected by the presence of DNA-PKcs. Gel filtration of Ku in the absence and the presence of DNA indicates that Ku does not form nonspecific aggregates. We conclude that, when bound to DNA, Ku is capable of self-association. These findings suggest that Ku binding at DNA DSBs will result in Ku self-association and a physical tethering of the broken DNA strands.