The synaptic complex of RecA protein participates in hybridization and inverse strand exchange reactions.

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.

Mechanism of aminoglycoside antibiotic kinase APH(3')-IIIa: role of the nucleotide positioning loop.

The aminoglycoside antibiotic resistance kinases (APHs) and the Ser/Thr/Tyr protein kinases share structural and functional homology but very little primary sequence conservation (<5%). A region of structural, but not amino acid sequence, homology is the nucleotide positioning loop (NPL) that closes down on the enzyme active site upon binding of ATP. This loop region has been implicated in facilitating phosphoryl transfer in protein kinases; however, there is no primary sequence conservation between APHs and protein kinases in the NPL. There is an invariant Ser residue in all APH NPL regions, however. This residue in APH(3')-IIIa (Ser27), an enzyme widespread in aminoglycoside-resistant Enterococci, Streptococci, and Staphylococci, directly interacts with the beta-phosphate of ATP through the Ser hydroxymethyl group and the amide hydrogen in the 3D structure of the enzyme. Mutagenesis of this residue to Ala and Pro supported a role for the Ser amide hydrogen in nucleotide capture and phosphoryl transfer. A molecular model of the proposed dissociative transition state, which is consistent with all of the available mechanistic data, suggested a role for the amide of the adjacent Met26 in phosphoryl transfer. Mutagenesis studies confirmed the importance of the amide hydrogen and suggest a mechanism where Ser27 anchors the ATP beta-phosphate facilitating bond breakage with the gamma-phosphate during formation of the metaphosphate-like transition, which is stabilized by interaction with the amide hydrogen of Met26. The APH NPL therefore acts as a lever, promoting phosphoryl transfer to the aminoglycoside substrate, with the biological outcome of clinically relevant antibiotic resistance.