CHARACTERIZATION OF 2 NUCLEAR MAMMALIAN HOMOLOGOUS DNA-PAIRING ACTIVITIES THAT DO NOT REQUIRE ASSOCIATED EXONUCLEASE ACTIVITY

We have developed an assay to study homologous DNA-pairing activities in mammalian nuclear extracts, This assay is derived from the POM blot assay, described earlier, which was specific for RecA activity in bac terial crude extracts. In the present work, proteins from mammalian nu clear extracts were resolved by electrophoresis on SDS/polyacrylamide gels and then electrotransferred onto a nitrocellulose membrane coated with Circular single-stranded DNA (ssDNA), The blot obtained was incu bated with a labeled homologous double-stranded DNA (dsDNA). Homologou s pairing between the ssDNA and the labeled dsDNA was detected by auto radiography as a radioactive spot on the membrane, In nuclear extracts from mammalian cells, we found two major polypeptides of 100 and 75 k Da, able to promote the formation of stable plectonemic joints, Joint molecule formation required at least one homologous end on the dsDNA, but either end of the dsDNA could be recruited to initiate the reactio n, For each polypeptide, the reaction required divalent cations such a s Mg2+, Ca2+, or Mn2+. Although ATP was not necessary, ADP was inhibit ory in each case, Unlike most of the known eukaryotic DNA-pairing prot eins, both activities identified here were able to promote the formati on of joint molecules without requiring an associated exonuclease acti vity, In addition, these two proteins were detected in cell lines from different tissues and from different mammalian species (human, mouse, and hamster).