The Synaptic Complex of RecA Protein Participates in Hybridization and Inverse Strand Exchange Reactions

Abstract

RecA protein catalyzes strand exchange between homologous single-stranded and double-stranded DNAs. In the presence of ATPgammaS, the post-strand exchange synaptic complex is a stable end product that can be studied. Here we ask whether such complexes can hybridize to or exchange with DNA, 2'-OMe RNA, PNA, or LNA oligonucleotides. Using a gel mobility shift assay, we show that the displaced strand of a 45 bp synaptic complex can hybridize to complementary oligonucleotides with different backbones to form a four-stranded (double D-loop) joint that survives removal of the RecA protein. This hybridization reaction, which confirms the single-stranded character of the displaced strand in a synaptic complex, might initiate recombination-dependent DNA replication if it occurs in vivo. We also show that either strand of the heteroduplex in a 30 bp synaptic complex can be replaced with a homologous DNA oligonucleotide in a strand exchange reaction that is mediated by the RecA filament. Consistent with the important role that deoxyribose plays in strand exchange, oligonucleotides with non-DNA backbones did not participate in this reaction. The hybridization and strand exchange reactions reported here demonstrate that short synaptic complexes are dynamic structures even in the presence of ATPgammaS.

RecA-mediated strand invasion of DNA by oligonucleotides substituted with 2-aminoadenine and 2-thiothymine

“…Unless otherwise indicated, the double-stranded targets were composed of regular bases. The design of paired pcONs suitable for use with RecA protein was guided by our earlier study of recombinase-mediated double D-loop formation using unmodified ONs (28). Incoming ONs, which were homologous to the double-stranded targets, had a DNA backbone that was at least 30 nt long.…”

“…First, the backbone prevented filament formation with recombinase (30), thereby ensuring unhindered access of the annealing ON to the synaptic complex. Second, it prevented dissociation of the double D-loop joint by blocking recombinase-mediated inverse strand exchange (28,31). Incoming and annealing ONs were designated as regular (rONs) or pseudo-complementary (pcONs) based on the absence or presence of nA and sT bases.…”

“…Unless otherwise noted, the three-step protocol described in Gamper et al (28) was followed. Briefly, a single-stranded incoming DNA 30-mer (0.8 pmol) was incubated for 10 min at 37°C with 24 pmol of RecA protein in 8 μl of SEB supplemented with 1 mM ATPγS unless otherwise specified.…”

“…Although RecA protein catalyzes D-loop formation in linear dsDNA, deproteinization leads to rapid branch migration and release of the ON. We have previously shown that the displaced strand in a RecA-stabilized D-loop can hybridize to a complementary ON provided that it has an RNA-like backbone and is shorter than the incoming DNA oligomer (28). This reaction leads to a four-stranded double D-loop joint that can survive deproteinization even when present in a linear dsDNA.…”

Nucleic Acid Electronics: from Chemistry to Circuits

“…Because PNA is initially neutral, perhaps this can be metallized in pH ranges that are more amenable to the use of RecA [ 76 ]. Apparently RecA-ssDNA strands can facilitate strand exchange in PNA and other nucleic acids as well [86]. In the event that researchers try to return to using PNA as the base material, based on these hopes, there are still multiple repercussions to that choice.…”