Characterisation of a DNA pairing activity copurifying with DNA ligase in a partially purified extract from rat testis

Abstract

Rat testicular nuclear extracts were fractionated sequentially on phosphocellulose, heparin-agarose and ssDNA-cellulose columns, in order to isolate and characterise a strand-transfer activity from a mammalian meiotic tissue. A partially purified fraction, eluting at 0.6 M KCl from ssDNA-cellulose column, catalyzed the formation of two classes of products migrating slowly on an agarose gel. The formation of one of these classes of products - the aggregates - was dependent on the presence of both the substrates (M13mp19 RF III and M13mp19 ssDNA) and on homology. The presence of ATP was essential for the formation of aggregates, though its hydrolysis was not required. EM analysis of the products indicated the presence of structures which resembled paired DNA molecules: duplex-duplex paired (Y-shaped and ds-ds paired structures) and ss-ds paired (duplex DNA paired with the single-stranded DNA) structures, indicating the presence of a pairing protein in the fraction. However, α- and σ-structures were not observed. The other class of products, seen as discrete bands, were identified biochemically and by electron microscopy as ligated products. A DNA ligase-adenylate adduct of molecular weight 100 kDa was formed by the fraction. Both 5' to 3' and 3' to 5' exonucleases were absent and hence did not contribute to the formation of the products.