Proc. Nati. Acad. Sci. USA Vol. 89, pp. 6492-6496, July 1992 Biochemistry
The synapsis event in the homologous pairing of DNAs: RecA recognizes and pairs less than one helical repeat of DNA mapping/triple-stranded DNA) PEGGY HSIEH, CAROL S. CAMERINIOTERO*, AND R. DANIEL CAMERINI-OTERO (homologous rmombinatlon/gene
Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
Communicated by Howard A. Nash, April 9, 1992 (received for review March S, 1992)
Davis. pBR322 and pUC18 plasmid DNAs and M13mpl8 replicative-form DNA were from Pharmacia. Oligonucleotides were synthesized and purified by passage over a Mono Q column (Pharmacia) (7). Oligonucleotides were 5'-endlabeled with [y-32P]ATP (New England Nuclear) and T4 polynucleotide kinase (Pharmacia) (7). Oligonucleotides homologous to pUC18 (see Fig. 2) spanned positions 230-285, 230-267, 230-255, and 230-249 (8). Oligonucleotides completely homologous to pBR322 (see Fig. 3) spanned positions 15-29, 10-29, and 4359-29 (9). The 20L series of oligonucleotides (see Fig. 4) had homology to pBR322 at positions 10-29, 20-29, 22-29, 24-29, and 26-29. The 20R series (see Fig. 4) had homology to positions 20-39, 20-29, 20-27, 20-25, and 20-23. DNA concentrations are expressed as moles of nucleotide or by weight. Synaptic-Complex Formation. Synaptic complexes were formed by incubating 1.8 1xM oligonucleotide (15 ng), 18 ILM duplex DNA (150 ng), and 1.5 ,uM RecA (1.5 pug) in 25 ,4 of 20 mM Tris-HCI, pH 7.5/0.4 mM dithiothreitol/12.5 mM MgCl2/0.3 mM ATP[IyS] (Fluka)/1.1 mM ADP (Sigma) for 15 min at 37C. Following synaptic complex formation, 10-20 units of the appropriate restriction endonuclease (New England Biolabs) was added and incubation continued for an additional 5 min. The reaction was quenched by the addition of SDS (1%) and EDTA (10 mM). Reaction mixtures were electrophoresed at 0.6 V/cm in 1% agarose gels in 40mM Tris acetate, pH 8.0/1 mM EDTA with ethidium bromide (1 ,ug/ml) for 14-16 hr at room temperature. Quantitation was determined by densitometer scanning of Polaroid 665 negatives. One hundred percent protection of the duplex corresponds to the duplex signal from 150 ng of duplex DNA incubated without restriction endonuclease minus the duplex signal from a control reaction containing duplex DNA, RecA, and the appropriate restriction endonuclease. In some cases, synaptic complex assays were quenched by the addition of 1% SDS and electrophoresed at 0.6 V/cm in 1% agarose gels in 89 mM Tris borate, pH 8.3/5 mM MgCl2 for 14-16 hr at room temperature. Deproteinized Joint Molecules. Synaptic complexes were formed as described above except that 5'-32P-labeled oligonucleotides were used. The reactions were quenched by the addition of SDS and EDTA. After electrophoresis and autoradiography, joint molecules were quantified by densitometric scanning.
A key step in homologous recombination is ABSTRACT the alignment and pairing of homologous DNAs. The Eschernchia coli RecA protein initiates iring by binding to singlestrand DNA, forming a helical nucleoprotein filament. We demonstrate that in the presence of the nonhydrolyzable ATP analogue adenosine 5'-[vthioJtrlphosphate and ADP, RecA can pair a homologous oligonucleotide 15 bases long with a duplex DNA to yield synaptic complexes consisting of the oligonucleotide and duplex DNA stabilized by RecA. RecA can pair as few as eight bases of homology to form such synaptic complexes. The homologous DNAs remain paired to each other upon removal of RecA provided that the length of shared homology is at least 26 base pairs. Based on our findings and the work of others, we propose that in vitro, one helical turn of a RecA nucleoprotein filament containing approximately six RecA monomers and 15 bases of single-strand DNA is the functional unit sufficient to carry out the homology search.
The essential problem in genetic recombination is how homologous DNA sequences find each other and align. Defining the basic pairing structure that carries out the homology search is critical for unraveling the mechanism of homologous pairing by recombinase proteins like the Escherichia coli RecA protein (reviewed in ref. 1). We reasoned that determination of the structure of strand-exchange intermediates might be facilitated by limiting pairing to very small regions. Recently, we described the stable pairing at the end of a linear duplex of very short regions of homology (
The synapsis event in the homologous pairing of DNAs: RecA recognizes and pairs less than one helical repeat of DNA mapping/triple-stranded DNA) PEGGY HSIEH, CAROL S. CAMERINIOTERO*, AND R. DANIEL CAMERINI-OTERO (homologous rmombinatlon/gene
Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
Communicated by Howard A. Nash, April 9, 1992 (received for review March S, 1992)
Davis. pBR322 and pUC18 plasmid DNAs and M13mpl8 replicative-form DNA were from Pharmacia. Oligonucleotides were synthesized and purified by passage over a Mono Q column (Pharmacia) (7). Oligonucleotides were 5'-endlabeled with [y-32P]ATP (New England Nuclear) and T4 polynucleotide kinase (Pharmacia) (7). Oligonucleotides homologous to pUC18 (see Fig. 2) spanned positions 230-285, 230-267, 230-255, and 230-249 (8). Oligonucleotides completely homologous to pBR322 (see Fig. 3) spanned positions 15-29, 10-29, and 4359-29 (9). The 20L series of oligonucleotides (see Fig. 4) had homology to pBR322 at positions 10-29, 20-29, 22-29, 24-29, and 26-29. The 20R series (see Fig. 4) had homology to positions 20-39, 20-29, 20-27, 20-25, and 20-23. DNA concentrations are expressed as moles of nucleotide or by weight. Synaptic-Complex Formation. Synaptic complexes were formed by incubating 1.8 1xM oligonucleotide (15 ng), 18 ILM duplex DNA (150 ng), and 1.5 ,uM RecA (1.5 pug) in 25 ,4 of 20 mM Tris-HCI, pH 7.5/0.4 mM dithiothreitol/12.5 mM MgCl2/0.3 mM ATP[IyS] (Fluka)/1.1 mM ADP (Sigma) for 15 min at 37C. Following synaptic complex formation, 10-20 units of the appropriate restriction endonuclease (New England Biolabs) was added and incubation continued for an additional 5 min. The reaction was quenched by the addition of SDS (1%) and EDTA (10 mM). Reaction mixtures were electrophoresed at 0.6 V/cm in 1% agarose gels in 40mM Tris acetate, pH 8.0/1 mM EDTA with ethidium bromide (1 ,ug/ml) for 14-16 hr at room temperature. Quantitation was determined by densitometer scanning of Polaroid 665 negatives. One hundred percent protection of the duplex corresponds to the duplex signal from 150 ng of duplex DNA incubated without restriction endonuclease minus the duplex signal from a control reaction containing duplex DNA, RecA, and the appropriate restriction endonuclease. In some cases, synaptic complex assays were quenched by the addition of 1% SDS and electrophoresed at 0.6 V/cm in 1% agarose gels in 89 mM Tris borate, pH 8.3/5 mM MgCl2 for 14-16 hr at room temperature. Deproteinized Joint Molecules. Synaptic complexes were formed as described above except that 5'-32P-labeled oligonucleotides were used. The reactions were quenched by the addition of SDS and EDTA. After electrophoresis and autoradiography, joint molecules were quantified by densitometric scanning.
A key step in homologous recombination is ABSTRACT the alignment and pairing of homologous DNAs. The Eschernchia coli RecA protein initiates iring by binding to singlestrand DNA, forming a helical nucleoprotein filament. We demonstrate that in the presence of the nonhydrolyzable ATP analogue adenosine 5'-[vthioJtrlphosphate and ADP, RecA can pair a homologous oligonucleotide 15 bases long with a duplex DNA to yield synaptic complexes consisting of the oligonucleotide and duplex DNA stabilized by RecA. RecA can pair as few as eight bases of homology to form such synaptic complexes. The homologous DNAs remain paired to each other upon removal of RecA provided that the length of shared homology is at least 26 base pairs. Based on our findings and the work of others, we propose that in vitro, one helical turn of a RecA nucleoprotein filament containing approximately six RecA monomers and 15 bases of single-strand DNA is the functional unit sufficient to carry out the homology search.
The essential problem in genetic recombination is how homologous DNA sequences find each other and align. Defining the basic pairing structure that carries out the homology search is critical for unraveling the mechanism of homologous pairing by recombinase proteins like the Escherichia coli RecA protein (reviewed in ref. 1). We reasoned that determination of the structure of strand-exchange intermediates might be facilitated by limiting pairing to very small regions. Recently, we described the stable pairing at the end of a linear duplex of very short regions of homology (
