To understand the molecular basis of gene targeting, we have studied interactions of nucleoprotein rfaments comprised of single-stranded DNA and RecA protein with chromatin templates reconstituted from linear duplex DNA and histones. We observed that for the chromatin templates with histone/DNA mass ratios of 0.8 and 1.6, the efficiency of homologous pairing was indistinguishable from that of naked duplex DNA but strand exchange was repressed. In contrast, the chromatin templates with a histone/DNA mass ratio of 9.0 supported neither homologous pairing nor strand exchange. The addition of histone H1, in stoichiometric amounts, to chromatin templates quells homologous pairing. The pairing of chromatin templates with nucleoprotein filaments of RecA protein-single-stranded DNA proceeded without the production of detectable networks of DNA, suggesting that coaggregates are unlikely to be the intermediates in homologous pairing. The application of these observations to strategies for gene targeting and their implications for models of genetic recombination are discussed.In recent years several laboratories have shown homologous recombination between DNA newly introduced into recipient mammalian cells and a target chromosomal gene. Such "gene targeting" has implications for the study of gene expression, the development of animal models for human genetic diseases, the improvement of livestock animals and plants, the production of products of pharmaceutical importance, and the repair of genetic defects (1, 2). Mouse embryonic stem cells have been used for gene targeting to create mutant genes and to correct mutant phenotypes (3-5). Thus, mammalian somatic cells do contain the enzymic machinery for homologous recombination (6-9). Capecchi (10) has shown that homologous recombination is maximal in S phase of the cell cycle and is manifested within 30 min after the DNA is injected into the nucleus.The frequency of the occurrence of recombinants has been low and variable, depending upon the cell type and the locus selected (2). A robust targeting system has not yet emerged. Efforts to develop such a system should examine the DNA at the locus of interest when it is in an active state and when it is in a repressed state. Indeed, it has been illustrated that the state of chromatin determines the accessibility of DNA to various enzymatic processes (11). A transcriptionally active gene shows greater sensitivity to nucleases than does the repressed gene. This repression in eukaryotes is believed to be exerted by histones, the ubiquitous general repressors (12).To investigate the molecular mechanisms ofgene targeting, we chose the Escherichia coli RecA protein as a model since the system is well defined at the genetic and molecular levels (13,14). With this system we have addressed two related issues: (i) what are the parameters of eukaryotic chromatin that determine the efficiency of gene targeting and (ii) how does chromatin structure influence homologous pairing and strand exchange of duplex DNA? EXPERIMENTAL PROCEDURES...
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