YqjH and YqjW are Bacillus subtilis homologs of the UmuC/DinB or Y superfamily of DNA polymerases that are involved in SOS-induced mutagenesis in Escherichia coli. While the functions of YqjH and YqjW in B. subtilis are still unclear, the comparisons of protein structures demonstrate that YqjH has 36% identity to E. coli DNA polymerase IV (DinB protein), and YqjW has 26% identity to E. coli DNA polymerase V (UmuC protein). In this report, we demonstrate that both YqjH and the products of the yqjW operon are involved in UV-induced mutagenesis in this bacterium. Furthermore, resistance to UV-induced damage is significantly reduced in cells lacking a functional YqjH protein. Analysis of stationary-phase mutagenesis indicates that absences of YqjH, but not that of YqjW, decreases the ability of B. subtilis to generate revertants at the hisC952 allele via this system. These data suggest a role for YqjH in the generation of at least some types of stationary-phase-induced mutagenesis.In Escherichia coli, the dinB gene is required for bacteriophage untargeted mutagenesis (UTM), an error-prone pathway observed when undamaged DNA infects SOS-induced E. coli cells (4, 55). Overexpression of the dinB gene confers a mutator phenotype on the cells (22). However, mutations in the dinB gene only caused a modest UV sensitivity phenotype, indicating that this gene product might not play a major role in the tolerance of DNA lesions introduced by UV irradiation into E. coli (22). The genetic requirements for UTM include the recA, uvrA, uvrB, uvrC, and polA genes, as well as DNA polymerase III (DNA Pol III), in addition to dinB (22,26). However, when the dinB gene is overexpressed on a multicopy plasmid, these requirements for genes besides dinB for UTM are bypassed (22). In 1999, it was discovered that the purified DinB protein has a template-directed, DNA-dependent DNA polymerase activity and it was designated the fourth DNA polymerase in E. coli (DNA Pol IV) (51).The DNA damage-inducible UmuDЈ and UmuC proteins are required for another type of SOS mutagenesis in E. coli (40). UmuCD-dependent translesion DNA synthesis allows cells to replicate past DNA damage-induced lesions that would normally block the continuing polymerization by the major replication DNA polymerase (DNA Pol III) in E. coli. This translesion synthesis results in an increased mutation rate (21, 42). The translesion DNA synthesis process requires the products of the SOS-regulated recA gene and the umuDC operon, which was originally identified by screening for E. coli mutants that were not mutable by UV light and other agents (21, 42). The umuDC gene products are also known to be essential components of chromosomal UTM (9, 27), a transient increase in the mutation frequency of chromosomal genes following induction of the SOS response (9, 27, 30). In 1999, UmuC or UmuDЈ 2 C was discovered to be a template-directed, DNAdependent DNA polymerase that was designated the fifth DNA polymerase in E. coli (DNA Pol V) (34, 49).It has very recently become apparent that UmuC ...

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The transcription factor SCL is critically required for the establishment of the hematopoietic lineage as well as for proper endothelial development, however it is not required for maintenance of HSCs or endothelial cells in the adult. Conflicting data exists regarding the developmental function of SCL, namely whether it acts as a master regulator, actively patterning mesoderm toward hematopoietic/endothelial development at the expense of other lineages, or is merely necessary to maintain the earliest committed hematopoietic and endothelial precursors. To answer this question in a mammalian model, we have engineered murine ES cells with a conditional doxycycline-inducible SCL transgene, and evaluated the effects of pulses of SCL expression at defined time points during in vitro development. During embryoid body differentiation, a pulse of SCL expression during mesodermal patterning results in enhanced hematopoiesis at later time points, as assayed by CFC activity and FACS analysis, as well significantly increasing the frequency of bipotent hematopoietic/endothelial hemangioblast CFCs. Concomitantly, SCL represses cardiac development, assayed by beating cardiomyocyte frequency, and molecular markers of the cardiac lineage. SCL also represses paraxial mesodermal developmental potential, severely reducing both the earliest PDGFαR+/Flk-1neg paraxial progenitors, as well as repressing levels of later markers including Pax3. Through chimeric embryoid bodies in which SCL cells are mixed with GFP-labelled ES cells, we show formally that this effect is cell-autonomous. Finally, we tested whether expression of SCL would enable hematopoiesis during ES cell monolayer differentiation. Hematopoiesis requires inter-germ layer inductive signaling which occurs in 3 dimensions but not in monolayer culture, thus ES cells do not spontaneously differentiate into blood in this 2 dimensional culture. We show that when mesoderm is produced during monolayer culture of wild-type ES cells, or control cells not expressing SCL, it is biased towards paraxial or cardiac differentiation. However when given a pulse of SCL expression, these pathways are repressed, PDGFαRneg/Flk-1+ presumptive lateral plate progenitors are produced, and abundant hematopoietic differentiation occurs. This data in the mammalian system thus strongly supports the former hypothesis, of an active role for SCL in mesodermal patterning.