DNA double-strand breaks may be induced by endonucleases, ionizing radiation, chemical agents, and mechanical forces or by replication of single-stranded nicked chromosomes. Repair of double-strand breaks can occur by homologous recombination or by nonhomologous end joining. A system was developed to measure the efficiency of plasmid gap repair by homologous recombination using either chromosomal or plasmid templates. Gap repair was biased toward gene conversion events unassociated with crossing over using either donor sequence. The dependence of recombinational gap repair on genes belonging to the RAD52 epistasis group was tested in this system. RAD51, RAD52, RAD57, and RAD59 were required for efficient gap repair using either chromosomal or plasmid donors. No homologous recombination products were recovered from rad52 mutants, whereas a low level of repair occurred in the absence of RAD51, RAD57, or RAD59. These results suggest a minor pathway of strand invasion that is dependent on RAD52 but not on RAD51. The residual repair events in rad51 mutants were more frequently associated with crossing over than was observed in the wild-type strain, suggesting that the mechanisms for RAD51-dependent and RAD51-independent events are different. Plasmid gap repair was reduced synergistically in rad51 rad59 double mutants, indicating an important role for RAD59 in RAD51-independent repair.Transformation of fungi with plasmid DNA has yielded important insights into the mechanisms of recombination and double-strand break repair (DSBR). Early studies by Hinnen et al. (17) provided evidence for integration of circular nonreplicating plasmids by homologous recombination into the yeast genome. In a subsequent study, Orr-Weaver et al. (38) elaborated more thoroughly the way in which circular and linear nonreplicating DNA molecules recombine with homologous chromosomal sequences. They showed that DNA ends are highly recombinogenic and interact directly with homologous sequences. If two restriction cuts are made within a plasmid region homologous to chromosomal DNA, thereby producing a double-strand gap, the resulting deleted linear plasmids transform at a high frequency and are faithfully repaired during the integration process. Using linear replicating plasmids, Orr-Weaver and Szostak (37) reported the recovery of approximately equal numbers of integrated and nonintegrated plasmids and concluded that gene conversion by double-strand gap repair can occur either with or without crossing over. These studies formed the basis for the DSBR model (65). They also observed circularization of linear plasmid DNA, suggesting the presence of additional, recombination-independent repair pathways. Subsequent studies of plasmid gap repair in Saccharomyces cerevisiae and Ustilago maydis indicated a lower association of crossing over with gene conversion (12,44). Studies in Drosophila and mouse cells have also shown a very low association of crossing over (Ͻ5%) during DSBR (35,48).When plasmids capable of autonomous replication are cut within reg...

Ad4BP, also known as SF-1, is a cell type-specific transcription factor regulating all the steroidogenic P-450 genes. Recently, the targeted disruption of the mouse ftz-f1 gene encoding Ad4BP/SF-1 has established its essential function in both adrenal and gonadal development and sexual differentiation. As an initial step toward understanding its role in the cascade of gene activations necessary for the differentiation of the steroidogenic tissues and the sex differentiation of the gonads, we isolated and characterized the rat ad4bp gene. A sequence analysis of the ad4bp gene revealed that another nuclear factor ELP was also transcribed from the same gene by alternative promoter usage and splicing. The promoter of the ad4bp gene showed activities in the steroidogenic cells such as Y-1 adrenocortical cells and I-10 testicular Leydig cells when examined by transient transfection assays. Using deletion analysis and site-directed mutagenesis, we identified a cis-element at the position from -82 bp to -77 bp in the 5'-upstream region. The cis-element was identical to the consensus E box element, which is the binding site for the basic-helix-loop-helix proteins. Gel mobility shift analyses revealed the amount of a binding factor to this E box in the nuclear extract prepared from the rat testes attained a maximal level 1 week after birth and then decreased dramatically thereafter, and only trace amounts were detected in adult rats. In contrast, the binding factor in the ovaries attained a maximal level just after birth and kept its level thereafter. These dimorphic expressions of the binding factor to the E box correlated well with those of Ad4BP, and thus suggested that the expression of Ad4BP, and thus suggested that the expression of Ad4BP is transcriptionally regulated through this E box element.

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