Characterization of Reca Mediated homologous pairing on nitrocellulose membrane.
Reactions between a single-stranded DNA (ssDNA) and a double-stranded DNA (dsDNA) provide an efficient model to study RecA promoted homologous recombination. We have devised an assay in which the ssDNA is first bound to a nitrocellulose membrane. RecA protein is loaded on this membrane (loading step) which is then incubated with a labelled homologous dsDNA (incubation step). Since this assay can be used for study of mutant RecA proteins or RecA-like activities in crude extracts from other organisms, we have characterized the reaction promoted on the membrane. Under these new conditions, the reaction keeps the main characteristics observed with classical assays performed in solution: increasing NaCl concentration destabilized the RecA-DNA complex, ATP gamma S was required for formation of stable RecA-DNA complex, initiation of the reaction exhibits the same polarity as in classical assays, a complete strand exchange with a 44 bp long duplex oligonucleotide has been recorded under our conditions. Moreover, our results indicate that the binding of RecA protein itself to the nitrocellulose membrane did not impair its ability to promote homologous pairing. Pairing reactions involving long dsDNA (6407 bp) were more efficient with hydrolysable ATP than with ATP gamma S only when the ssDNA was bound to the membrane. Furthermore, ATP hydrolysis was not required when using short dsDNA (44 bp). These results constitute experimental support for a new role for the ATPase activity of RecA protein: the energy produced could favor the initiation of RecA mediated recombination involving long stretches of DNA which have restricted freedom to rotation.
Bertrand
P
P
Section de Biologie, Institut Curie, Paris, France.
Akhmedov
A T
AT
Lopez
B S
BS
D000255:Adenosine Triphosphate / Q000378:metabolism; D017104:Bacteriophage M13 / Q000235:genetics; D001483:Base Sequence; D003101:Collodion; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism*; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D011938:Rec A Recombinases / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D008567:Membranes, Artificial; D000255:Adenosine Triphosphate; D003101:Collodion; D011938:Rec A Recombinases