The RAD51 gene of Saccharomyces cerevisiae encodes a RecA like protein, which is involved in the recombinational repair of double strand breaks. We have isolated the RAD51 homologue, rhp51+, of the distantly related yeast strain Schizosaccharomyces pombe by heterologous hybridization. DNA sequence analysis of the rhp51+ gene revealed an open reading frame of 365 amino acids. Comparison of the amino acid sequences of RAD51 and rhp51+ showed a high level of conservation: 69% identical amino acids. There are two Mlul sites in the upstream region which may be associated with cell cycle regulation of the rhp51+ gene. The rhp51+ null allele, constructed by disruption of the coding region, is extremely sensitive to X-rays, indicating that the rhp51+ gene, like RAD51, is also involved in the repair of X-ray damage. The structural and functional homology between rhp51+ and RAD51 suggests evolutionary conservation of certain steps in the recombinational repair pathway.

The Schizosaccharomyces pombe rhp51+, rad22+ and rhp54+ genes are homologous to RAD51, RAD52 and RAD54 respectively, which are indispensable in the recombinational repair of double-strand breaks (DSBs) in Saccharomyces cerevisiae. The rhp51Delta and rhp54Delta strains are extremely sensitive to ionizing radiation; the rad22Delta mutant turned out to be much less sensitive. Homologous recombination in these mutants was studied by targeted integration at the leu1-32 locus. These experiments revealed that rhp51Delta and rhp54Delta are equally impaired in the integration of plasmid molecules (15-fold reduction), while integration in the rad22Delta mutant is only reduced by a factor of two. Blot-analysis demonstrated that the majority of the leu+ transformants of the wild-type and rad22Delta strains have integrated one or more copies of the vector. Gene conversion events were observed in less than 10% of the transformants. Interestingly, the relative contribution of gene conversion events is much higher in a rhp51Delta and a rhp54Delta background. Meiotic recombination is hardly affected in the rad22Delta mutant. The rhp51Delta and rhp54Delta strains also show minor deficiencies in this type of recombination. The viability of spores is 46% in the rad22Delta strain and 27% in the rhp54Delta strain, as compared with wild-type cells. However, in the rhp51Delta mutant the spore viability is only 1.7%, suggesting an essential role for Rhp51 in meiosis. The function of Rhp51 and Rhp54 in damage repair and recombination resembles the role of Rad51 and Rad54 in S. cerevisiae. Compared with Rad52 from S. cerevisiae, Rad22 has a much less prominent role in the recombinational repair pathway in S. pombe.

The RAD52 gene of Saccharomyces cerevisiae is required for recombinational repair of double-strand breaks. Using degenerate oligonucleotides based on conserved amino acid sequences of RAD52 and rad22, its counterpart from Schizosaccharomyces pombe, RAD52 homologs from man and mouse were cloned by the polymerase chain reaction. DNA sequence analysis revealed an open reading frame of 418 amino acids for the human RAD52 homolog and of 420 amino acid residues for the mouse counterpart. The identity between the two proteins is 69% and the overall similarity 80%. The homology of the mammalian proteins with their counterparts from yeast is primarily concentrated in the N-terminal region. Low amounts of RAD52 RNA were observed in adult mouse tissues. A relatively high level of gene expression was observed in testis and thymus, suggesting that the mammalian RAD52 protein, like its homolog from yeast, plays a role in recombination. The mouse RAD52 gene is located near the tip of chromosome 6 in region G3. The human equivalent maps to region p13.3 of chromosome 12. Until now, this human chromosome has not been implicated in any of the rodent mutants with a defect in the repair of double-strand breaks.

Endogenous glucocorticoids (GC), which are under control of the hypothalamic-pituitary-adrenal axis, play an important role in controlling chronic inflammatory demyelinating diseases, like multiple sclerosis (MS). Increased hypothalamic-pituitary-adrenal axis activity has been found in MS patients and appeared to be negatively associated with acute inflammation. Exogenous GC are frequently used to treat relapses in MS, but the response to this treatment differs among patients, suggesting differences in sensitivity to GC. Previous, relatively small studies investigating GC sensitivity have yielded conflicting results. In the present study, we have investigated GC sensitivity in peripheral blood cells of MS patients (n = 117) and healthy controls (n = 45). GC sensitivity was measured by the in vitro suppressive effect of GC on lipopolysaccharide-stimulated TNF-alpha production. Blood cells of MS patients, especially relapsing remitting MS patients, were less sensitive to GC compared with blood cells of healthy controls. This turned out to be unrelated to previous treatment with exogenous GC expressed as frequency of courses of iv steroids or interval since last course. The use of interferon beta was found to be associated with a lower GC sensitivity. However, after correction for the use of interferon beta, relapsing remitting MS patients remained less sensitive to GC.

The RAD54 gene of Saccharomyces cerevisiae encodes a putative helicase, which is involved in the recombinational repair of DNA damage. The RAD54 homologue of the fission yeast Schizosaccharomyces pombe, rhp54+, was isolated by using the RAD54 gene as a heterologous probe. The gene is predicted to encode a protein of 852 amino acids. The overall homology between the mutual proteins of the two species is 67% with 51% identical amino acids and 16% similar amino acids. A rhp54 deletion mutant is very sensitive to both ionizing radiation and UV. Fluorescence microscopy of the rhp54 mutant cells revealed that a large portion of the cells are elongated and occasionally contain aberrant nuclei. In addition, FACS analysis showed an increased DNA content in comparison with wild-type cells. Through a minichromosome-loss assay it was shown that the rhp54 deletion mutant has a very high level of chromosome loss. Furthermore, the rhp54 mutation in either a rad17 or a cdc2.3w mutant background (where the S-phase/mitosis checkpoint is absent) shows a significant reduction in viability. It is hypothesized that the rhp54+ gene is involved in the recombinational repair of UV and X-ray damage and plays a role in the processing of replication-specific lesions.