Abstract
Under conditions that diminish secondary structure in single-stranded DNA, stable presynaptic filaments can be formed by recA protein in the presence of the nonhydrolyzable analog ATP gamma S, without the need for Escherichia coli single strand binding protein. Such stable presynaptic filaments resemble those formed in the presence of ATP and pair efficiently with homologous duplex DNA. Since this kind of stable filament does not displace a strand from the duplex molecule, it provides a model substrate to study synapsis independent of the earlier and later stages of the recA reaction. Even though detectable strand displacement did not occur in the presence of ATP gamma S, both single strand and double strand breaks in duplex DNA stimulated homologous pairing. These and related observations support the view that the presynaptic nucleoprotein filament and naked duplex DNA intertwine to form a nascent joint in which the duplex DNA is partially unwound, i.e. in which the pitch of the involved duplex segment is reduced.
Highlights
From the Demrtments of H u m n Genetics and Molecular Biophysicsand Biochemistry, Yale Uniuersity School of Medicine, New Hauen,’Connecticut06510
Single-stranded recA NucleoproteinFilaments Madewith ATPyS Efficiently Paired with Homologous Duplex DNAThe following experiments tested the hypothesis that, even in the presence of ATP+, SSB is dispensable as a cofactor for recA protein if conditions areused that diminish secondary structure insingle-stranded DNA
When recA protein is preincubated with single strands in 12 mM MgC12instead of 1 mM MgCl, secondary structure in the DNA presents an obstacle to the formation of active presynaptic complexes (Muniyappa et at., 1984)
Summary
Networks within which homologous alignment occurs, probably by facilitated diffusion (Gonda and Radding, 1983; Gonda et al, 1985; Tsang et al, 1985a). In the experiments described here, preparations of linear or supercoiled DNA that were passed through amock nicking procedure (performed without restriction enzyme), or that were reisolated from a low melting point agarose gel, or both, yielded reactions identical to those obtained with duplex DNAs that had not been so treated. Strand exchange was monitored by two methods: by measuring the amount of the linear duplex DNA which becomes sensitive to SI nuclease as a result of the displacement of one of its strands (Wu etal., 1983),or by detecting the appearance of the final product of strand exchange, nicked circular duplex, after separatingthe DNA species by agarosegel electrophoresis (Coxand Lehman, 1981a).We verified that, under the conditions of the SIassay, recA protein in the presence of ATPyS does not interfere with digestion of single-stranded DNA. The buffer used for microinjection was mM Tris (pH7.5), 1mM MgC12,and thespecimens were negativelystained with 1%uranyl formate
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