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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 bacterial crude extracts. In the present work, proteins from mammalian nuclear 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 incubated with a labeled homologous double-stranded DNA (dsDNA). Homologous pairing between the ssDNA and the labeled dsDNA was detected by autoradiography as a radioactive spot on the membrane. In nuclear extracts from mammalian cells, we found two major polypeptides of 100 and 75 kDa, 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 reaction. For each polypeptide, the reaction required divalent cations such as Mg2+, Ca2+, or Mn2+. Although ATP was not necessary, ADPwas inhibitory in each case. Unlike most of the known eukaryotic DNA-pairing proteins, both activities identified here were able to promote the formation of joint molecules without requiring an associated exonuclease activity. In addition, these two proteins were detected in cell lines from different tissues and from different mammalian species (human, mouse, and hamster). The most-documented process involving search for homology and homologous DNA pairing is homologous recombination. Besides recombination, homologous pairing could be involved in other fundamental biological processes such as chromosome pairing (for review see ref. 1), gene inactivation (for review see ref. 2), initiation of viral replication (3), and DNA repair. Base pairing is also involved in spliceosome assembly, which may result in formation of a Holliday-like structure (4). The first DNA pairing and strand exchange protein to be discovered was the Escherichia coli RecA protein. In vivo, the involvement of RecA protein in mutagenesis and recombination is well documented. In vitro, RecA protein is able to align one single-stranded DNA (ssDNA) with a double-stranded DNA (dsDNA) and to promote formation of stable plectonemic joints in a sequence-dependent manner (for review see refs. 5-7). Recently, Rad5l, a protein involved in DNA repair and recombination in Saccharomyces cerevisiae, has been shown to promote homologous pairing and strand exchange (8). Pairing and strand exchange proteins have been identified in virus-infected cells (9-13) and in many eukaryotic organisms (14-21). Unlike RecA protein, the three best-characterized eukaryotic proteins-human HPP-1 (21), yeast SEP1/ STP,B (14,15), and Drosophila Rrpl (19) proteins-contain an The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. intrinsic or tightly associated exonuclease activity which is thought to be required for the initial synapsis step. However, when resected dsDNA is used or when an exonuclease activity is present in the preparation, the classical three-strand reaction can lead to artifacts, as discussed elsewhere (22). Thus, demonstration of mammalian proteins able to promote homologous pairing without such requirement of associated exonuclease activity is of particular interest. We have developed an in vitro assay to measure homologous pairing reaction: the pairing on membrane (POM) blot. This assay was tested with pure RecA protein from E. coli (23). In the present work, we have adapted the POM blot to the analysis of pairing activities in mammalian nuclear extracts. MATERIALS AND METHODS Reagents. Nitrocellulose membranes (HybondC-super; 0.45-,um pores) were purchased from Amersham. dsand ssDNA-cellulose was purchased from Sigma. ATP, ADP, adenosine 5'-[y-thio]triphosphate (ATP['yS]), dNTPs, protease inhibitors, proteinase K, and DNA restriction and modification enzymes were purchased from Boehringer Mannheim. Cells. HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and glutamine. Cells were checked for the absence of mycoplasma contamination by Hoechst 33342 fluorescence. DNA. Wild-type bacteriophage M13 or pBSTII phagemid (Stratagene) dsand ssDNAs were prepared as described (24). dsDNA was labeled with 32p, filling in the 5' protruding ends by using polymerase I large fragment, as described (24). In some experiments 5' ends of the dsDNA were labeled by using polynucleotide kinase and ['y-32P]ATP. Preparation of Nuclei and Nuclear Extracts. Nuclei from 2-5 x 108 cells were isolated by hypotonic lysis and nuclear extracts were prepared as described previously (25). Cytoplasmic S100 fraction was prepared as described (26). POM Assay. Protein samples without prior boiling were electrophoretically resolved on SDS/10% polyacrylamide gels (27). After electrophoresis, gels were equilibrated for 20 min at room temperature in transfer buffer (25 mM Tris/192 mM glycine, pH 8.3/0.05% SDS). The protein bands were then electrophoretically transferred onto nitrocellulose membrane coated with ssDNA prepared as described (23). The membrane was incubated in preincubation buffer (33 mM Tris HCl, pH 7.5/2 mM dithiothreitol/1 mM MgCl2/5% instant nonfat Abbreviations: ssDNA, single-stranded DNA; dsDNA, doublestranded DNA; POM, pairing on membrane; POMpn, n-kDa POM protein; ATP[yS], adenosine 5'-[y-thio]triphosphate. *On leave from: St. Petersburg Nuclear Physics Institute, Gatchina, Russia. tPresent address: Schleicher & Schuell, BP 32, ZAI du Petit Parc, 5 rue des Fontenelles, 78920 Ecquevilly, France. ITo whom reprint requests should be addressed.