Genetic variance modifies apoptosis susceptibility in mature oocytes via alterations in DNA repair capacity and mitochondrial ultrastructure.

Although the identification of specific genes that regulate apoptosis has been a topic of intense study, little is known of the role that background genetic variance plays in modulating cell death. Using germ cells from inbred mouse strains, we found that apoptosis in mature (metaphase II) oocytes is affected by genetic background through at least two different mechanisms. The first, manifested in AKR/J mice, results in genomic instability. This is reflected by numerous DNA double-strand breaks in freshly isolated oocytes, causing a high apoptosis susceptibility and impaired embryonic development following fertilization. Microinjection of Rad51 reduces DNA damage, suppresses apoptosis and improves embryonic development. The second, manifested in FVB mice, results in dramatic dimorphisms in mitochondrial ultrastructure. This is correlated with cytochrome c release and a high apoptosis susceptibility, the latter of which is suppressed by pyruvate treatment, Smac/DIABLO deficiency, or microinjection of 'normal' mitochondria. Therefore, background genetic variance can profoundly affect apoptosis in female germ cells by disrupting both genomic DNA and mitochondrial integrity.

Homologous pairing promoted by the human Rad52 protein.

The Rad52 protein, which is unique to eukaryotes, plays important roles in the Rad51-dependent and the Rad51-independent pathways of DNA recombination. In the present study, we have biochemically characterized the homologous pairing activity of the HsRad52 protein (Homo sapiens Rad52) and found that the presynaptic complex formation with ssDNA is essential in its catalysis of homologous pairing. We have identified an N-terminal fragment (amino acid residues 1-237, HsRad52(1-237)) that is defective in binding to the human Rad51 protein, which catalyzed homologous pairing as efficiently as the wild type HsRad52. Electron microscopic visualization revealed that HsRad52 and HsRad52(1-237) both formed nucleoprotein filaments with single-stranded DNA. These lines of evidence suggest the role of HsRad52 in the homologous pairing step of the Rad51-independent recombination pathway. Our results reveal the striking similarity between HsRad52 and the Escherichia coli RecT protein, which functions in a RecA-independent recombination pathway.

Homologous-pairing activity of the human DNA-repair proteins Xrcc3.Rad51C.

The human Xrcc3 protein is involved in the repair of damaged DNA through homologous recombination, in which homologous pairing is a key step. The Rad51 protein is believed to be the only protein factor that promotes homologous pairing in recombinational DNA repair in mitotic cells. In the brain, however, Rad51 expression is extremely low, whereas XRCC3, a human homologue of Saccharomyces cerevisiae RAD57 that activates the Rad51-dependent homologous pairing with the yeast Rad55 protein, is expressed. In this study, a two-hybrid analysis conducted with the use of a human brain cDNA library revealed that the major Xrcc3-interacting protein is a Rad51 paralog, Rad51C/Rad51L2. The purified Xrcc3.Rad51C complex, which shows apparent 1:1 stoichiometry, was found to catalyze the homologous pairing. Although the activity is reduced, the Rad51C protein alone also catalyzed homologous pairing, suggesting that Rad51C is a catalytic subunit for homologous pairing. The DNA-binding activity of Xrcc3.Rad51C was drastically decreased in the absence of Xrcc3, indicating that Xrcc3 is important for the DNA binding of Xrcc3.Rad51C. Electron microscopic observations revealed that Xrcc3.Rad51C and Rad51C formed similar filamentous structures with circular single-stranded DNA.

Human Rad51 amino acid residues required for Rad52 binding.

The Rad51 protein, a homologue of the bacterial RecA protein, is an essential factor for both meiotic and mitotic recombination. The N-terminal domain of the human Rad51 protein (HsRad51) directly interacts with DNA. Based on a yeast two-hybrid analysis, it has been reported that the N-terminal region of the Saccharomyces cerevisiae Rad51 protein binds Rad52;S. cerevisiae Rad51 and Rad52 both activate the homologous pairing and strand exchange reactions. Here, we show that the HsRad51 N-terminal region, which corresponds to the Rad52-binding region of ScRad51, does not exhibit strong binding to the human Rad52 protein (HsRad52). To investigate its function, the C-terminal region of HsRad51 was randomly mutagenized. Although this region includes the two segments corresponding to the putative DNA-binding sites of RecA, all seven of the mutants did not decrease, but instead slightly increased, the DNA binding. In contrast, we found that some of these HsRad51 mutations significantly decreased the HsRad52 binding. Therefore, we conclude that these amino acid residues are required for the HsRad51.HsRad52 binding. HsRad52, as well as S. cerevisiae Rad52, promoted homologous pairing between ssDNA and dsDNA, and higher homologous pairing activity was observed in the presence of both HsRad51 and HsRad52 than with either HsRad51 or HsRad52 alone. The HsRad51 F259V mutation, which strongly impaired the HsRad52 binding, decreased the homologous pairing in the presence of both HsRad51 and HsRad52, without affecting the homologous pairing by HsRad51 alone. This result suggests the importance of the HsRad51.HsRad52 interaction in homologous pairing.