The SOS response in bacteria includes a global transcriptional response to DNA damage. DNA damage is sensed by the highly conserved recombination protein RecA, which facilitates inactivation of the transcriptional repressor LexA. Inactivation of LexA causes induction (derepression) of genes of the LexA regulon, many of which are involved in DNA repair and survival after DNA damage. To identify potential RecA-LexAregulated genes in Bacillus subtilis, we searched the genome for putative LexA binding sites within 300 bp upstream of the start codons of all annotated open reading frames. We found 62 genes that could be regulated by putative LexA binding sites. Using mobility shift assays, we found that LexA binds specifically to DNA in the regulatory regions of 54 of these genes, which are organized in 34 putative operons. Using DNA microarray analyses, we found that 33 of the genes with LexA binding sites exhibit RecA-dependent induction by both mitomycin C and UV radiation. Among these 33 SOS genes, there are 22 distinct LexA binding sites preceding 18 putative operons. Alignment of the distinct LexA binding sites reveals an expanded consensus sequence for the B. subtilis operator: 5-CGAACATATGTTCG-3. Although the number of genes controlled by RecA and LexA in B. subtilis is similar to that of Escherichia coli, only eight B. subtilis RecA-dependent SOS genes have homologous counterparts in E. coli.Exposure of prokaryotes to DNA-damaging agents results in the induction of a diverse set of physiological responses collectively called the SOS response (8, 55). As first characterized in Escherichia coli, the SOS response includes an enhanced capacity for recombinational repair, enhanced capacity for excision repair, enhanced mutagenesis (due to error-prone repair), and inhibition of cell division (i.e., filamentation). Induction of the SOS response is due to the coordinate derepression of a number of SOS or din (for damage-inducible) genes. The SOS response to DNA damage in Bacillus subtilis is similar to that of E. coli (26,56,58), but unlike E. coli, the B. subtilis SOS system is also induced in competent cells in the absence of any DNA-damaging treatment (25, 57, 58). As in E. coli, SOS gene expression in B. subtilis is controlled by two proteins (which are themselves products of SOS genes): the LexA protein (also called DinR) (40,54), which represses the transcription of din genes by binding to the SOS operator (31), and the RecA protein (30), which is activated by single-stranded DNA (29,42) to stimulate the proteolytic autodigestion of LexA (24,31). Thus, an SOS gene is defined by two criteria-RecA-dependent induction by DNA damage and a binding site for LexA overlapping its promoter.By contrast with E. coli, where more than 30 SOS genes have been identified (7,8), only 5 B. subtilis SOS genes have been shown to meet both SOS gene criteria thus far: recA, lexA, uvrB (formerly dinA), dinB, and dinC (also called tagC) (4,9,15,25).

Chemical genomics involves generating large collections of small molecules and using them to modulate cellular states. Despite recent progress in the systematic synthesis of structurally diverse compounds, their use in screens of cellular circuitry is still an ad hoc process 1-4 . Here, we outline a general, efficient approach called gene expression-based highthroughput screening (GE-HTS) in which a gene expression signature is used as a surrogate for cellular states, and we describe its application in a particular setting: the identification of compounds that induce the differentiation of acute myeloid leukemia cells. In screening 1,739 compounds, we identified 8 that reliably induced the differentiation signature and, furthermore, yielded functional evidence of bona fide differentiation. The results indicate that GE-HTS may be a powerful, general approach for chemical screening.To prove the feasibility of GE-HTS, we applied the method to the identification of compounds capable of inducing terminal differentiation of acute myelogenous leukemia (AML) cells. The plausibility of differentiation induction in leukemia is suggested by a rare subtype of AML known as acute promyelocytic leukemia (APL) in which treatment with all-trans retinoic acid (ATRA) results in clinical remissions through modulation of a mutated retinoic acid receptor alpha 5,6 . Unfortunately, ATRA has no clinical efficacy in other subtypes of AML 7 . But the fact that all forms of AML show a block of differentiation suggests that differentiation therapy might be possible, provided that the right compounds could be identified. The mechanism underlying such blocked differentiation is unknown in most cases; thus, it is not possible to carry out small-molecule screens against a validated target protein. Instead, a cell-based screening approach is needed. The usual approach to assaying the myeloid differentiation phenotype, however, involves a combination of visual inspection of nuclear morphology and biochemical tests, such as nitroblue tetrazolium (NBT) reduction 8 , neither of which readily lend themselves to a highthroughput screening platform. We therefore sought to use GE-HTS to identify compounds capable of inducing the differentiation program in AML without first knowing the crucial targets of this process.The first step in the GE-HTS procedure is defining the gene expression signatures of the biological states of interest. To accomplish this, we carried out oligonucleotide microarray-based gene expression profiling of pretreatment bone marrow samples derived from individuals affected with AML, as well as of fully differentiated peripheral blood neutrophils and monocytes derived from unaffected donors (gene expression data are available in Supplementary Table 1a,b online). We thereby identified genes correlated with the neutrophil-versus-AML or monocyte-versus-AML distinctions (Fig. 1). We selected from these differentiation-correlated genes a handful of marker genes that represented the diversity of myeloid differentiation yet could be easil...

The SRS2 (Suppressor of RAD Six screen mutant 2) gene encodes an ATP-dependent DNA helicase that regulates homologous recombination in Saccharomyces cerevisiae. Mutations in SRS2 result in a hyper-recombination phenotype, sensitivity to DNA damaging agents and synthetic lethality with mutations that affect DNA metabolism. Several of these phenotypes can be suppressed by inactivating genes of the RAD52 epistasis group that promote homologous recombination, implicating inappropriate recombination as the underlying cause of the mutant phenotype. Consistent with the genetic data, purified Srs2 strongly inhibits Rad51-mediated recombination reactions by disrupting the Rad51-ssDNA presynaptic filament. Srs2 interacts with Rad51 in the yeast two-hybrid assay and also in vitro. To investigate the functional relevance of the Srs2-Rad51 complex, we have generated srs2 truncation mutants that retain full ATPase and helicase activities, but differ in their ability to interact with Rad51. Importantly, the srs2 mutant proteins attenuated for Rad51 interaction are much less capable of Rad51 presynaptic filament disruption. An internal deletion in Srs2 likewise diminishes Rad51 interaction and anti-recombinase activity. We also present evidence that deleting the Srs2 C-terminus engenders a hyper-recombination phenotype. These results highlight the importance of Rad51 interaction in the anti-recombinase function of Srs2, and provide evidence that this Srs2 function can be uncoupled from its helicase activity.