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.
Gamper
Howard B
HB
Division of Hematology/Oncology, University of Pennsylvania School of Medicine, BRB II/III Room 713, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104, USA. hbgamper@mail.med.upenn.edu
Nulf
Christopher J
CJ
Corey
David R
DR
Kmiec
Eric B
EB
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005914:Globins / Q000737:chemistry / Q000235:genetics; D006801:Humans; D019868:Kanamycin Kinase / Q000737:chemistry / Q000235:genetics; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry*; D015347:RNA Probes / Q000737:chemistry; D011938:Rec A Recombinases / Q000737:chemistry*; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid
D004277:DNA, Single-Stranded; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D015347:RNA Probes; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D005914:Globins; D019868:Kanamycin Kinase; D011938:Rec A Recombinases