Amino acid sequence analysis has established that the homologous pairing protein of Ustilago maydis, known previously in the literature as recl, is encoded by REC2, a gene essential for recombinational repair and meiosis with regional homology to Escherichia coli RecA. The 70-kDa recl protein is most likely a proteolytic degradation product of REC2, which has a predicted mass of 84 kDa but which runs anomalously during sodium dodecyl sulfate-gel electrophoresis with an apparent mass of 110 kDa. To facilitate purification of the protein product, the REC2 gene was overexpressed from a vector that fused a hexahistidine leader sequence onto the amino terminus, enabling isolation of the REC2 protein on an immobilized metal alfinity column. The purified protein exhibits ATP-dependent DNA renaturation and DNA-dependent ATPase activities, which were reactions characteristic of the protein as purified from cell extracts of U. maydis. Homologous pairing activity was established in an assay that measures recognition via non-Watson-Crick bonds between identical DNA strands. A size threshold of about 50 bp was found to govern pairing between linear duplex molecules and homologous single-stranded circles. Joint molecule formation with duplex DNA well under the size threshold was efficiently catalyzed when one strand of the duplex was composed of RNA. Linear duplex molecules with hairpin caps also formed joint molecules when as few as three RNA residues were present.Genetic recombination can be envisioned as a pathway that proceeds stepwise through a search for sequence homology, DNA pairing, heteroduplex formation, and exchange of strands. Elucidation of the process has come in part from detailed analysis of Escherichia coli RecA protein. Studies on the biochemical mechanism have revealed the protein to catalyze homologous pairing of DNA molecules in a reaction that transduces the energy of nucleotide cofactor binding to changing conformational states of the protein and to promote strand exchange by coupling nucleotide hydrolysis to unidirectional processing of the crossed-strand recombination intermediate (22,24,25,28,38). The RecA protein has become the paradigm for thinking about the mechanism of DNA pairing in general homologous recombination. The ubiquity of RecA in bacteria has reinforced the hope that lessons learned from studies of this prototype system may be extended beyond the realm of prokaryotes.Evidence supporting the notion that eukaryotes might conduct recombination through a RecA-like DNA pairing mechanism has come from two approaches. Genetic studies led to the discovery, first made in Saccharomyces cerevisiae (1,2,5,14,30) and later in other eukaryotes, of proteins homologous to RecA (for a review, see reference 11). On the other hand, biochemical studies with Ustilago maydis revealed the presence of an activity that could promote a number of DNA pairing reactions, some of which resembled reactions catalyzed by RecA protein (15). These included ATP-stimulated reassociation of complementary single strands, u...

Cultured tobacco (Nicotiana tabacum, var Xanthi) cells contain a topoisomerase that removes positive and negative supercoils from DNA. The enzyme has an estimated molecular mass of 30,000 daltons under denaturing conditions, but may exist as a multimeric protein in the native state. Activity is enhanced significantly by either MgCI2 or CaC12, but other divalent cations are much less effective in stimulating DNA relaxation. The purified enzyme acts by altering the linking number in topological steps of one and is inhibited by berenil or camptothecin, not novobiocin. Taken together, these data identify this enzyme as a type I topoisomerase.DNA topoisomerases play a crucial role in many cellular processes including replication, transcription, and recombination ( tein-DNA intermediate and does not require an energy cofactor. These reaction parameters define Int as a type I topoisomerase (see 11 for review). Additional information outlining the important role that other topoisomerases play in recombination has been provided by Chiba et al. (2). In that study, it was demonstrated that purified T4 DNA topoisomerase promotes illegitimate recombination.Recently, Christman et al. (3) reported that yeast strains containing topo 1,2 mutants have much higher levels of mitotic recombination. This enhancement was rather specific for rDNA rearrangement in the nucleolus. In support of these data is the observation that the rDNA spacer contains at least three DNA sequences, which are preferentially identified by type I DNA topoisomerase as a site of action (18). The catalytic rate of DNA relaxation at these sites exceeds interaction at nonspecific sites by three orders of magnitude. As suggested by Petes and Fink (13), the control oftopoisomerase activity during recombination may be essential in order to regulate the development of large inversions, deletions, or translocations.We have been interested in the mechanism of genetic recombination in plants. We chosen cultured tobacco cells as our primary source of enzyme and set out to purify the DNA topoisomerases from these cells. In this report, we describe the isolation and purification of a type I topoisomerase from cultured tobacco cells. In addition, we present the biochemical characterization of the enzyme and suggest possible roles for type I topoisomerase in the process of genetic recombination.

To gain a more complete understanding of the process of in vitro chromatin assembly, an examination of the energy requirements of nucleosome formation must be undertaken. The experiments outlined in this manuscript address this issue by making use of the Xenopus laevis S-150 cell-free extract. The S-150 catalyzes chromatin assembly on circular DNA templates dependent either on the exogenous addition of ATP or regeneration of endogenous ATP. We define two distinct, but temporally ordered, phases of the overall process. The first, nucleosome formation, occurs in the presence of endogenous levels of ATP, while the second phase, chromatin assembly, which we define as the development of properly spaced nucleosomes, requires a higher level of ATP. Both phases lead to a distribution of molecules with similar superhelical densities. Taken together, these data suggest that chromatin assembly may consist of two distinct steps differing in their strategy cofactor requirement. The experiments presented in this manuscript support the concept that nucleosomes first assemble, perhaps randomly, on the DNA and are gradually matured into a canonical chromatin structure with periodic spacing.

Amino acid sequence analysis has established that the homologous pairing protein of Ustilago maydis, known previously in the literature as rec1, is encoded by REC2, a gene essential for recombinational repair and meiosis with regional homology to Escherichia coli RecA. The 70-kDa rec1 protein is most likely a proteolytic degradation product of REC2, which has a predicted mass of 84 kDa but which runs anomalously during sodium dodecyl sulfate-gel electrophoresis with an apparent mass of 110 kDa. To facilitate purification of the protein product, the REC2 gene was overexpressed from a vector that fused a hexahistidine leader sequence onto the amino terminus, enabling isolation of the REC2 protein on an immobilized metal affinity column. The purified protein exhibits ATP-dependent DNA renaturation and DNA-dependent ATPase activities, which were reactions characteristic of the protein as purified from cell extracts of U. maydis. Homologous pairing activity was established in an assay that measures recognition via non-Watson-Crick bonds between identical DNA strands. A size threshold of about 50 bp was found to govern pairing between linear duplex molecules and homologous single-stranded circles. Joint molecule formation with duplex DNA well under the size threshold was efficiently catalyzed when one strand of the duplex was composed of RNA. Linear duplex molecules with hairpin caps also formed joint molecules when as few as three RNA residues were present.

The role of DNA topoisomerases in plant cell metabolism is currently under investigation in our laboratory. Using a purified type I topoisomerase from cultured tobacco, we have carried out a biochemical characterization of enzymatic behavior. The enzyme relaxes negatively supercoiled DNA in the presence of MgCl2, and to a lesser extent in the presence of KCl. Phosphorylation of the topoisomerase does not influence its activity and it is not stimulated by the presence of histones H1 or H5. The enzyme may act in either a processive or distributive manner depending on reaction conditions. The anti-tumor drug, camptothecin, induces significant breakage by the enzyme on purified DNA molecules unless destabilized by the addition of KCl. The tobacco topoisomerase I can catalyze the formation of stable nucleosomes on circular DNA templates, suggesting a role for the enzyme in chromatin assembly.