Journal of Molecular Biology
Volume 227, Issue 1, 5 September 1992, Pages 334-346
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Article
Structural data suggest that the active and inactive forms of the RecA filament are not simply interconvertible

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Abstract

We have used electron microscopy to examine the two major conformational states of the helical filament formed by the RecA protein of Escherichia coli. The compressed filament, formed in the absence of a nucleotide cofactor either as a self-polymer or on a single-stranded DNA molecule, is characterized in solution by about 6·1 subunits per turn of a 76 Å pitch helix, and appears to be inactive with respect to all RecA activity. The active state of the filament, formed with ATP or an ATP analog on either a single or double-stranded DNA substrate, has about 6·2 subunits per turn of a 94 Å pitch helix. Measurements of the contour length of RecA-covered single-stranded DNA circles in ice, formed in the absence of nucleotide cofactor, indicate that each RecA subunit binds five bases, in contrast to the three bases or base-pairs per subunit in the active state. The different stoichiometries of DNA binding suggests that the two polymeric forms are not interconvertible, as has been suggested on biochemical grounds. A three-dimensional reconstruction of the inactive state shows the same general features as the 83 Å pitch filament present in the RecA crystal. This structural similarity and the fact that the crystal does not contain ATP or DNA suggests that the crystal structure is more similar to the compressed filament than the active, extended filament.

References (32)

Cited by (135)

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    The electron microscopic [7,8] and crystallographic studies of E. coli RecA [9–11] together with high-resolution cryo-electron microscopy studies of Rad51 [12,13] have revealed a remarkably conserved structure of a RecA/Rad51 nucleoprotein filament [4–6]. The ssDNA in the nucleoprotein filament is extended 1.5-fold compared to the canonical B-form DNA with a binding stoichiometry of 3 nucleotides/RecA monomer [7,8,11]. The triplet in ssDNA serves as a fundamental unit of recognition of homology, pairing and strand exchange with homologous duplex DNA [4–6,11,14].

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    RecA monomers cooperatively polymerize onto ssDNA to form a right-handed helix around the ssDNA with ATP cofactors bound between the monomers (Fig. 2A; [74,75]). The DNA in those filaments is approximately 150% of the length of corresponding B-form DNA [76–80]. RecA binds to the ssDNA with an exact stoichiometry of three nucleotides per RecA monomer (Fig. 2B).

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This work was supported by NIH grant GM35269.

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