ABSTRACT
BACKGROUND: Homologous recombination is of eminent importance both in germ cells, to generate genetic diversity during meiosis, and in somatic cells, to safeguard DNA from genotoxic damage. The genetically well-defined RAD52 pathway is required for these processes in the yeast Saccharomyces cerevisiae. Genes similar to those in the RAD52 group have been identified in mammals. It is not known whether this conservation of primary sequence extends to conservation of function. RESULTS: Here we report the isolation of cDNAs encoding a human and a mouse homolog of RAD54. The human (hHR54) and mouse (mHR54) proteins were 48% identical to Rad54 and belonged to the SNF2/SW12 family, which is characterized by amino-acid motifs found in DNA-dependent ATPases. The hHR54 gene was mapped to chromosome 1p32, and the hHR54 protein was located in the nucleus. We found that the levels of hHR54 mRNA increased in late G1 phase, as has been found for RAD54 mRNA. The level of mHR54 mRNA was elevated in organs of germ cell and lymphoid development and increased mHR54 expression correlated with the meiotic phase of spermatogenesis. The hHR54 cDNA could partially complement the methyl methanesulfonate-sensitive phenotype of S. cerevisiae rad54 delta cells. CONCLUSIONS: The tissue-specific expression of mHR54 is consistent with a role for the gene in recombination. The complementation experiments show that the DNA repair function of Rad54 is conserved from yeast to humans. Our findings underscore the fundamental importance of DNA repair pathways: even though they are complex and involve multiple proteins, they seem to be functionally conserved throughout the eukaryotic kingdom.
Subject(s)
Conserved Sequence , DNA Repair , Nuclear Proteins/genetics , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Animals , Base Sequence , COS Cells , Chromosome Mapping , DNA Helicases , DNA Repair Enzymes , DNA, Complementary , DNA-Binding Proteins , Fungal Proteins/genetics , Gene Expression , Genetic Complementation Test , HeLa Cells , Humans , Mice , Molecular Sequence Data , Nuclear Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sequence Homology, Amino AcidABSTRACT
Gene conversion was first defined in yeast as a type of homologous recombination in which the donor sequence does not change. In chicken B cells, gene conversion builds the antigen receptor repertoire by introducing sequence diversity into the immunoglobulin genes. Immunoglobulin gene conversion continues at high frequency in an avian leukosis virus induced chicken B cell line. This cell line can be modified by homologous integration of transfected DNA constructs offering a model system for studying gene conversion in higher eukaryotes. In search for genes which might participate in chicken immunoglobulin gene conversion, we have identified chicken counterparts of the yeast RAD51, RAD52, and RAD54 genes. Disruption and overexpression of these genes in the chicken B cell line may clarify their role in gene conversion and gene targeting.
Subject(s)
Chickens/genetics , DNA Repair , Gene Conversion , Genes, Immunoglobulin , Recombination, Genetic , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Animals , Antibody Diversity , Avian Proteins , B-Lymphocytes/physiology , DNA Helicases , DNA Repair Enzymes , DNA-Binding Proteins/physiology , Epistasis, Genetic , Fungal Proteins/physiology , Immunoglobulin Light Chains/genetics , Mice , Molecular Sequence Data , Proteins/physiology , Rad51 Recombinase , Rad52 DNA Repair and Recombination Protein , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
Degenerate oligonucleotides encoding conserved regions of the Rad52 protein of S. cerevisiae and its homologue, the Rad22 protein of S. pombe, were used to clone a chicken RAD52 counterpart by the polymerase chain reaction. Sequence comparison of the chicken and yeast proteins reveals a strongly conserved region between positions 40 and 178 of the chicken Rad52 sequence indicating that this part of the protein is under strong evolutionary pressure. The first 39 amino acids and the 3' end of the chicken Rad52 homologue does not share significant similarity with the yeast proteins. High abundance of the mRNA in testis makes it likely that the chicken Rad52 protein plays a role in meiotic recombination.
