Double-strand DNA break (DSB) repair by homologous recombination occurs through the RAD52 pathway in Saccharomyces cerevisiae. Its biological importance is underscored by the conservation of many RAD52 pathway genes, including RAD54, from fungi to humans. We have analyzed the phenotype of mouse RAD54-/- (mRAD54-/-) cells. Consistent with a DSB repair defect, these cells are sensitive to ionizing radiation, mitomycin C, and methyl methanesulfonate, but not to ultraviolet light. Gene targeting experiments demonstrate that homologous recombination in mRAD54-/- cells is reduced compared to wild-type cells. These results imply that, besides DNA end-joining mediated by DNA-dependent protein kinase, homologous recombination contributes to the repair of DSBs in mammalian cells. Furthermore, we show that mRAD54-/- mice are viable and exhibit apparently normal V(D)J and immunoglobulin class-switch recombination. Thus, mRAD54 is not required for the recombination processes that generate functional immunoglobulin and T cell receptor genes.

Transcription‐coupled repair (TCR) is a universal sub‐pathway of the nucleotide excision repair (NER) system that is limited to the transcribed strand of active structural genes. It accomplishes the preferential elimination of transcription‐blocking DNA lesions and permits rapid resumption of the vital process of transcription. A defect in TCR is responsible for the rare hereditary disorder Cockayne syndrome (CS). Recently we found that mutations in the ERCC6 repair gene, encoding a putative helicase, underly the repair defect of CS complementation group B. Here we report the cloning and characterization of the Saccharomyces cerevisiae homolog of CSB/ERCC6, which we designate RAD26. A rad26 disruption mutant appears viable and grows normally, indicating that the gene does not have an essential function. In analogy with CS, preferential repair of UV‐induced cyclobutane pyrimidine dimers in the transcribed strand of the active RBP2 gene is severely impaired. Surprisingly, in contrast to the human CS mutant, yeast RAD26 disruption does not induce any UV‐, cisPt‐ or X‐ray sensitivity, explaining why it was not isolated as a mutant before. Recovery of growth after UV exposure was somewhat delayed in rad26. These findings suggest that TCR in lower eukaryotes is not very important for cell survival and that the global genome repair pathway of NER is the major determinant of cellular resistance to genotoxicity.

The highly conserved Saccharomyces cerevisiae Rad51 protein plays a central role in both mitotic and meiotic homologous DNA recombination. Seven members of the Rad51 family have been identified in vertebrate cells, including Rad51, Dmc1, and five Rad51-related proteins referred to as Rad51 paralogs, which share 20 to 30% sequence identity with Rad51. In chicken B lymphocyte DT40 cells, we generated a mutant with RAD51B/ RAD51L1, a member of the Rad51 family, knocked out. RAD51B؊/؊ cells are viable, although spontaneous chromosomal aberrations kill about 20% of the cells in each cell cycle. Rad51B deficiency impairs homologous recombinational repair (HRR), as measured by targeted integration, sister chromatid exchange, and intragenic recombination at the immunoglobulin locus. RAD51B؊/؊ cells are quite sensitive to the cross-linking agents cisplatin and mitomycin C and mildly sensitive to ␥-rays. The formation of damage-induced Rad51 nuclear foci is much reduced in RAD51B ؊/؊ cells, suggesting that Rad51B promotes the assembly of Rad51 nucleoprotein filaments during HRR. These findings show that Rad51B is important for repairing various types of DNA lesions and maintaining chromosome integrity. Double-strand DNA breaks (DSBs) occur during DNA replication and are produced by ionizing radiation. Since DSBs are so deleterious to the cell, it is not surprising that there are two DSB repair pathways: nonhomologous end joining (NHEJ) and homologous recombination repair (HRR). Repair of DSBs by HRR requires the presence of homologous duplex DNA elsewhere in the genome, i.e., either a homologous chromosome or, more likely, a sister chromatid. NHEJ simply acts to process and ligate broken ends without a requirement for extensive homology. These pathways are conserved from the yeast Saccharomyces cerevisiae to humans (5,8,9,19,49,53,64). While HRR is the primary mechanism of DSB repair in yeast, vertebrate cells use both the NHEJ and HRR pathways extensively (28,34,35,44). The analysis of radiosensitive yeast mutants has revealed a number of genes involved in HRR, which comprise the RAD52 epistasis group (reviewed in references 4, 29, and 51).Among the members of the RAD52 epistasis group, the structure and function of Rad51 have been conserved to a remarkable degree among all eukaryotes. Rad51 is structurally and functionally related to the Escherichia coli recombination protein RecA (reviewed in reference 32). The functional forms of both RecA and Rad51 are multimeric helical nucleoprotein filaments that form on single-stranded DNA ends produced at DSBs (41). These filaments are involved in the search for homologous sequence, DNA pairing, and strand exchange. Recombination intermediates produced in this way are then processed further in reactions that involve DNA synthesis, branch migration, resolution of Holliday junctions, and ligation (reviewed in reference 4). The conservation of the RAD52 epistasis group genes from yeast to vertebrate cells suggests that the basic mechanism of HRR is maintained during evolution. Howe...

Bone quality is an important determinant of osteoporosis, and proper osteoblast differentiation plays an important role in the control and maintenance of bone quality. We investigated the impact of activin signaling on human osteoblast differentiation, extracellular matrix formation, and mineralization. Activins belong to the transforming growth factor-beta superfamily and activin A treatment strongly inhibited mineralization in osteoblast cultures, whereas the activin antagonist follistatin increased mineralization. Osteoblasts produced activin A and follistatin in a differentiation-dependent manner, leading to autocrine regulation of extracellular matrix formation and mineralization. In addition, mineralization in a vascular smooth muscle cell-based model for pathological calcification was inhibited. Comparative activin A and follistatin gene expression profiling showed that activin signaling changes the expression of a specific range of extracellular matrix proteins prior to the onset of mineralization, leading to a matrix composition with reduced or no mineralizing capacity. These findings demonstrate the regulation of osteoblast differentiation and matrix mineralization by the activin A-follistatin system, providing the possibility to control bone quality as well as pathological calcifications such as atherosclerosis by using activin A, follistatin, or analogs thereof.

Error-free repair by homologous recombination of DNA double-strand breaks induced by ionizing radiation (IR) requires the Rad52 group proteins, including Rad51 and Rad54, in the yeast Saccharomyces cerevisiae [1]. The formation of a 'joint' molecule between the damaged DNA and the homologous repair template is a key step in recombination mediated by Rad51 and stimulated by Rad54 [2] [3] [4] [5]. Mammalian homologs of Rad51 and Rad54 have been identified [2] [3] [6]. Here, we demonstrate that mouse Rad54 (mRad54) formed IR-induced nuclear foci that colocalized with mRad51. Interaction between mRad51 and mRad54 was induced by genotoxic stress, but only when lesions that required mRad54 for their repair were formed. Interestingly, mRad54 was essential for the formation of IR-induced mRad51 foci. Rad54 belongs to the SWI2/SNF2 protein family, members of which modulate protein-DNA interactions in an ATP-driven manner [7]. Results of a topological assay suggested that purified human Rad54 (hRad54) protein can unwind double-stranded (ds) DNA at the expense of ATP hydrolysis. Unwinding of the homologous repair template could promote the formation or stabilization of hRad51-mediated joint molecules. Rad54 appears to be required downstream of other Rad52 group proteins, such as Rad52 and the Rad55-Rad57 heterodimer, that assist Rad51 in interacting with the broken DNA [2] [3] [4].

Cerebral palsy due to perinatal injury to cerebral white matter is usually not caused by genetic mutations, but by ischemia and/or inflammation. Here, we describe an autosomal-recessive type of tetraplegic cerebral palsy with mental retardation, reduction of cerebral white matter, and atrophy of the cerebellum in an inbred sibship. The phenotype was recorded and evolution followed for over 20 years. Brain lesions were studied by diffusion tensor MR tractography. Homozygosity mapping with SNPs was performed for identification of the chromosomal locus for the disease. In the 14 Mb candidate region on chromosome 7q22, RNA expression profiling was used for selecting among the 203 genes in the area. In postmortem brain tissue available from one patient, histology and immunohistochemistry were performed. Disease course and imaging were mostly reminiscent of hypoxic-ischemic tetraplegic cerebral palsy, with neuroaxonal degeneration and white matter loss. In all five patients, a donor splice site pathogenic mutation in intron 14 of the AP4M1 gene (c.1137+1G-->T), was identified. AP4M1, encoding for the mu subunit of the adaptor protein complex-4, is involved in intracellular trafficking of glutamate receptors. Aberrant GluRdelta2 glutamate receptor localization and dendritic spine morphology were observed in the postmortem brain specimen. This disease entity, which we refer to as congenital spastic tetraplegia (CST), is therefore a genetic model for congenital cerebral palsy with evidence for neuroaxonal damage and glutamate receptor abnormality, mimicking perinatally acquired hypoxic-ischemic white matter injury.

Refractive errors are the most common ocular disorders worldwide and may lead to blindness. Although this trait is highly heritable, identification of susceptibility genes has been challenging. We conducted a genome-wide association study for refractive error in 5,328 individuals from a Dutch population-based study with replication in four independent cohorts (combined 10,280 individuals in the replication stage). We identified a significant association at chromosome 15q14 (rs634990, P = 2.21 × 10−14). The odds ratio of myopia compared to hyperopia for the minor allele (minor allele frequency = 0.47) was 1.41 (95% CI 1.16–1.70) for individuals heterozygous for the allele and 1.83 (95% CI 1.42–2.36) for individuals homozygous for the allele. The associated locus is near two genes that are expressed in the retina, GJD2 and ACTC1, and appears to harbor regulatory elements which may influence transcription of these genes. Our data suggest that common variants at 15q14 influence susceptibility for refractive errors in the general population.

Ionizing radiation and interstrand DNA crosslinking compounds provide important treatments against cancer due to their extreme genotoxicity for proliferating cells. Both the efficacies of such treatments and the mutagenic potential of these agents are modulated by the ability of cells to repair the inflicted DNA damage. Here we demonstrate that homologous recombination-deficient mRAD54(-/-) mice are hypersensitive to ionizing radiation at the embryonic but, unexpectedly, not at the adult stage. However, at the adult stage mRAD54 deficiency dramatically aggravates the ionizing radiation sensitivity of severe combined immune deficiency (scid) mice that are impaired in DNA double-strand break repair through DNA end-joining. In contrast, regardless of developmental stage, mRAD54(-/-) mice are hypersensitive to the interstrand DNA crosslinking compound mitomycin C. These results demonstrate that the two major DNA double-strand break repair pathways in mammals have overlapping as well as specialized roles, and that the relative contribution of these pathways towards repair of ionizing radiation-induced DNA damage changes during development of the animal.