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. 1998 Sep 15;95(19):11071-6.
doi: 10.1073/pnas.95.19.11071.

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

T Nishinaka  1 , A Shinohara, Y Ito, S Yokoyama, T Shibata
Affiliations

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

T Nishinaka et al. Proc Natl Acad Sci U S A. .

Abstract

Escherichia coli RecA is a representative of proteins from the RecA family, which promote homologous pairing and strand exchange between double-stranded DNA and single-stranded DNA. These reactions are essential for homologous genetic recombination in various organisms. From NMR studies, we previously reported a novel deoxyribose-base stacking interaction between adjacent residues on the extended single-stranded DNA bound to RecA protein. In this study, we found that the same DNA structure was induced by the binding to Saccharomyces cerevisiae Rad51 protein, indicating that the unique DNA structure induced by the binding to RecA-homologs was conserved from prokaryotes to eukaryotes. On the basis of this structure, we have formulated the structure of duplex DNA within filaments formed by RecA protein and its homologs. Two types of molecular structures are presented. One is the duplex structure that has the N-type sugar pucker. Its helical pitch is approximately 95 A (18.6 bp/turn), corresponding to that of an active, or ATP-form of the RecA filament. The other is one that has the S-type sugar pucker. Its helical pitch is approximately 64 A (12.5 bp/turn), corresponding to that of an inactive, or ADP-form of the RecA filament. During this modeling, we found that the interconversion of sugar puckers between the N-type and the S-type rotates bases horizontally, while maintaining the deoxyribose-base stacking interaction. We propose that this base rotation enables base pair switching between double-stranded DNA and single-stranded DNA to take place, facilitating homologous pairing and strand exchange. A possible mechanism for strand exchange involving DNA rotation also is discussed.

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Figures

Figure 1
Figure 1
TRNOE spectra of d(TGACAT) bound to S. cerevisiae Rad51 protein or E. coli RecA protein in the presence of ATPγS. (a and b) d(TGACAT) bound to Rad51 protein. The 0.71 mM d(TGACAT), 67 μM S. cerevisiae Rad51 protein, 0.71 mM ATPγS, 20 mM d11-Tris⋅Cl (pH* 7.1), and 6.7 mM MgCl2 in D2O at 303 K. (c and d) d(TGACAT) bound to RecA protein. The 1.1 mM d(TGACAT), 54 μM E. coli RecA protein, 1.1 mM ATPγS, 20 mM d11-Tris⋅Cl (pH* 7.1), and 6.7 mM MgCl2 and 150 mM NaCl in D2O at 310 K. Mixing time of both spectra is 200 msec. The dotted lines indicate sequential connectivities of d(TGACAT). Signals around 4.7 ppm (indicated by an arrow in b) are derived from residual water.
Figure 2
Figure 2
Molecular models of the N-type (Left) and S-type (Center) duplex DNA structure within RecA protein filaments and B-form DNA (Right). The values of their pitches are set to 95 Å (18.6 bp/turn) for the N-type structure and 64 Å (12.5 bp/turn) for the S-type structure to satisfy those of active and inactive forms of RecA filaments, respectively. B-form DNA has a helical pitch of ≈36 Å (10.5 bp/turn). Each DNA molecule contains 18 base pairs. Note that both N-type and S-type duplex are extended by 1.5 times compared with B-form DNA but have different helical pitches. The N-type duplex DNA structure has a broad and open minor groove so that the single-stranded DNA can approach the duplex without severe steric hindrance.
Figure 3
Figure 3
Base rotation by interconversion of sugar puckers. (a) Top view of the base rotation caused by interconversion of the sugar puckers. The sugar pucker of the 5′-residue (T, top) is in the S-type (Left) and the N-type (Right), whereas that of the 3′-residue (A, bottom) is fixed in the S-type. Note that the hydrogen-bonding vector is rotated toward its major groove by the conversion from the S-type to the N-type. (b) Two types of deoxyribose-base stacking. All residues are in the S-type sugar pucker (Left) or the N-type (Right) sugar pucker.
Figure 4
Figure 4
A three-stranded DNA model for homology search and strand exchange considering the N–S interconversion of sugar puckers. (a) A molecular model of base pair switch between single- and double-stranded DNA. The bottom three residues are in the N-type and the top three residues are in the S-type. Note that the base pairing is altered by the conversion of sugar puckers between the N- and S-type. (b) Base rotation schemes for base pair switching against the interconversion of the sugar puckers. The bases are rotated toward the minor groove when the sugar puckers are converted from the N-type (Left) to the S-type (Right) and toward the major groove with the opposite (S-type to N-type) conversion.
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