RESUMEN
A 246-bp imperfect palindrome has the potential to form hairpin structures in single-stranded DNA during replication. Genetic evidence suggests that these structures are converted to double-strand breaks by the SbcCD nuclease and that the double-strand breaks are repaired by recombination. We investigated the role of a range of recombination mutations on the viability of cells containing this palindrome. The palindrome was introduced into the Escherichia coli chromosome by phage lambda lysogenization. This was done in both wt and sbcC backgrounds. Repair of the SbcCD-induced double-strand breaks requires a large number of proteins, including the components of both the RecB and RecF pathways. Repair does not involve PriA-dependent replication fork restart, which suggests that the double-strand break occurs after the replication fork has passed the palindrome. In the absence of SbcCD, recombination still occurs, probably using a gap substrate. This process is also PriA independent, suggesting that there is no collapse of the replication fork. In the absence of RecA, the RecQ helicase is required for palindrome viability in a sbcC mutant, suggesting that a helicase-dependent pathway exists to allow replicative bypass of secondary structures.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/genética , Conformación de Ácido Nucleico , Recombinación Genética , Proteínas Bacterianas/metabolismo , Bacteriófago lambda/genética , Replicación del ADN , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/metabolismo , Proteína de Replicación ARESUMEN
Urine is produced in the kidney by excretory nephrons and is drained by a tree-like system of collecting ducts to the ureter. The collecting ducts develop by arborisation of an initially unbranched epithelial rudiment, the ureteric bud, which ramifies through the surrounding mesenchyme and induces the formation of nephrons by mesenchyme-to-epithelial transition. The question of how collecting duct morphogenesis is controlled is an important one, from the points of view of both basic developmental biology and congenital renal pathology (multi- and polycystic renal disease, and some forms of renal agenesis, arise from defective collecting duct development). We report that neurturin, a neurotrophin related to glial cell line-derived neurotrophic factor and expressed in the developing kidney, acts as a collecting duct morphogen in culture. Applied in culture medium, it promotes epithelial branching and can induced branch initiation that has otherwise been blocked by depleting cultured kidneys of their sulfated proteoglycans or by antibody treatments. Applied locally on agarose beads, neurturin induces supernumerary ureteric buds to emerge from the wolffian duct and causes nearby collecting duct branches to distend to an abnormally large diameter. Like its receptors, neurturin is expressed by the developing collecting ducts themselves, suggesting that it forms an autocrine morphoregulatory control loop. This is in marked contrast to previously identified morphogens such as glial cell line derived neurotrophic factor and hepatocyte growth factor, which act in a paracrine manner.