Purification and characterization of a protein from human cells which promotes homologous pairing of DNA.
A human protein of approximately 120 kilodaltons has been purified to homogeneity based on its ability to catalyze the homology-dependent transfer of the complementary strand from a linear duplex DNA to a circular single-strand DNA. The activity was purified from an immature T-cell acute leukemic tumor cell line, with the majority of enrichment obtained by chromatography on a novel Z-DNA affinity column. The human homologous pairing protein was found to absolutely require homologous DNA substrates in a reaction that needs nearly stoichiometric amounts of protein. The homologous pairing activity is not stimulated by addition of exogenous ATP; however, the photo-cross-linking ATP analog 8-azidoadenosine 5'-[32P] triphosphate (8-N3-[32P]ATP) binds specifically to the homologous pairing protein. Electron microscopic analysis demonstrated the formation of all expected products. Intermediate strand-exchange products were shown to conserve the displaced DNA strands, eliminating many alternate explanations for the homologous pairing activity. These and other biochemical properties described in this report suggest that the nature of homologous pairing by the human protein is functionally similar to that of the bacterial RecA protein, although the exact mechanism of strand exchange may be somewhat different.
Moore
S P
SP
Laboratory of Chromosome Biology, National Cancer Institute-Frederick Cancer Research Facility, Maryland 21701.
Fishel
R
R
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels / Q000378:metabolism; D001386:Azides / Q000378:metabolism; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism*; D004270:DNA, Circular / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D006801:Humans; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000378:metabolism; D008854:Microscopy, Electron; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D011938:Rec A Recombinases / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D001386:Azides; D004270:DNA, Circular; D004277:DNA, Single-Stranded; D011506:Proteins; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D011938:Rec A Recombinases