Radiation sensitivity of the gastrula-stage embryo: Chromosome aberrations and mutation induction in lacZ transgenic mice: The roles of DNA double-strand break repair systems.

At the gastrula phase of development, just after the onset of implantation, the embryo proper is characterized by extremely rapid cell proliferation. The importance of DNA repair is illustrated by embryonic lethality at this stage after ablation of the genes involved. Insight into mutation induction is called for by the fact that women often do not realize they are pregnant, shortly after implantation, a circumstance which may have important consequences when women are subjected to medical imaging using ionizing radiation.

Rad51C is essential for embryonic development and haploinsufficiency causes increased DNA damage sensitivity and genomic instability.

Homologous recombination is essential for repair of DNA interstrand cross-links and double-strand breaks. The Rad51C protein is one of the five Rad51 paralogs in vertebrates implicated in homologous recombination. A previously described hamster cell mutant defective in Rad51C (CL-V4B) showed increased sensitivity to DNA damaging agents and displayed genomic instability.

The effects of MSH2 deficiency on spontaneous and radiation-induced mutation rates in the mouse germline.

Mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germline of mismatch repair deficient Msh2 knock-out mice. Spontaneous mutation rates in homozygous Msh2(-/-) males were significantly higher than those in isogenic wild-type (Msh2(+/+)) and heterozygous (Msh2(+/-)) mice. In contrast, the irradiated Msh2(-/-) mice did not show any detectable increases in their mutation rate, whereas significant ESTR mutation induction was observed in the irradiated Msh2(+/+) and Msh2(+/-) animals.

Cellular characterization of cells from the Fanconi anemia complementation group, FA-D1/BRCA2.

Fanconi anemia (FA) is an inherited cancer-susceptibility disorder, characterized by genomic instability and hypersensitivity to DNA cross-linking agents. The discovery of biallelic BRCA2 mutations in the FA-D1 complementation group allows for the first time to study the characteristics of primary BRCA2-deficient human cells. FANCD1/BRCA2-deficient fibroblasts appeared hypersensitive to mitomycin C (MMC), slightly sensitive to methyl methane sulfonate (MMS), and like cells derived from other FA complementation groups, not sensitive to X-ray irradiation.

Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions.

The BRCA2 tumor suppressor plays an important role in the repair of DNA damage by homologous recombination, also termed homology-directed repair (HDR). Human BRCA2 is 3,418 aa and is composed of several domains. The central part of the protein contains multiple copies of a motif that binds the Rad51 recombinase (the BRC repeat), and the C terminus contains domains that have structural similarity to domains in the ssDNA-binding protein replication protein A (RPA).

Chinese hamster cell mutant, V-C8, a model for analysis of Brca2 function.

The previously described Chinese hamster cell mutant V-C8 that is defective in Brca2 shows a very complex phenotype, including increased sensitivity towards a wide variety of DNA damaging agents, chromosomal instability, abnormal centrosomes and impaired formation of Rad51 foci in response to DNA damage. Here, we demonstrate that V-C8 cells display biallelic nonsense mutations in Brca2, one in exon 15 and the other in exon 16, both resulting in truncated Brca2 proteins. We generated several independent mitomycin C (MMC)-resistant clones from V-C8 cells that had acquired an additional mutation leading to the restoration of the open reading frame of one of the Brca2 alleles.

Inactivation of RAD52 aggravates RAD54 defects in mice but not in Schizosaccharomyces pombe.

RAD52 and RAD54 genes from Saccharomyces cerevisiae are required for double-strand break repair through homologous recombination and show epistatic interactions i.e., single and double mutant strains are equally sensitive to DNA damaging agents.

Germline mutation rates at tandem repeat loci in DNA-repair deficient mice.

Mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germline of non-exposed and irradiated severe combined immunodeficient (scid) and poly(ADP-ribose) polymerase (PARP-1-/-) deficient male mice. Non-exposed scid and PARP-/- male mice showed considerably elevated ESTR mutation rates, far higher than those in wild-type isogenic mice and other inbred strains. The irradiated scid and PARP-1-/- male mice did not show any detectable increases in their mutation rate, whereas significant ESTR mutation induction was observed in the irradiated wild-type isogenic males.

Function of DNA-protein kinase catalytic subunit during the early meiotic prophase without Ku70 and Ku86.

All components of the double-stranded DNA break (DSB) repair complex DNA-dependent protein kinase (DNA-PK), including Ku70, Ku86, and DNA-PK catalytic subunit (DNA-PKcs), were found in the radiosensitive spermatogonia. Although p53 induction was unaffected, spermatogonial apoptosis occurred faster in the irradiated DNA-PKcs-deficient scid testis. This finding suggests that spermatogonial DNA-PK functions in DNA damage repair rather than p53 induction.

DNA double-strand breaks and gamma-H2AX signaling in the testis.

Within minutes of the induction of DNA double-strand breaks in somatic cells, histone H2AX becomes phosphorylated at serine 139 and forms gamma-H2AX foci at the sites of damage. These foci then play a role in recruiting DNA repair and damage-response factors and changing chromatin structure to accurately repair the damaged DNA. These gamma-H2AX foci appear in response to irradiation and genotoxic stress and during V(D)J recombination and meiotic recombination.

Enhanced recovery of radiation-induced translocations from spermatogonial stem cells of p53 null mice.

X-ray-induced (4Gy) chromosomal translocations were studied in mouse spermatogonial stem cells with different p53 status by meiotic analysis at the spermatocyte stage, many cell generations after the moment irradiation. The results show enhanced recovery of translocations from p53 -/- mice relative to +/- and +/+ littermates. The enhanced recovery is probably due to an altered cell cycle distribution of the stem cells in the -/- mice leading to less radioresistant G(0)-G(1) transition cells, rather than differences in apoptotic response.

Mammalian Rad51C contributes to DNA cross-link resistance, sister chromatid cohesion and genomic stability.

The eukaryotic Rad51 protein is a structural and functional homolog of Escherichia coli RecA with a role in DNA repair and genetic recombination. Five paralogs of Rad51 have been identified in vertebrates, Rad51B, Rad51C, Rad51D, Xrcc2 and Xrcc3, which are also implicated in recombination and genome stability. Here, we identify a mammalian cell mutant in Rad51C.

Long-term effects of irradiation before adulthood on reproductive function in the male rhesus monkey.

Today, many patients, who are often young, undergo total body irradiation (TBI) followed by bone marrow transplantation. This procedure can have serious consequences for fertility, but the long-term intratesticular effects of this treatment in primates have not yet been studied. Testes and epididymides of rhesus monkeys that received doses of 4-8.

Brca2 (XRCC11) deficiency results in radioresistant DNA synthesis and a higher frequency of spontaneous deletions.

We show here that the radiosensitive Chinese hamster cell mutant (V-C8) of group XRCC11 is defective in the breast cancer susceptibility gene Brca2. The very complex phenotype of V-C8 cells is complemented by a single human chromosome 13 providing the BRCA2 gene, as well as by the murine Brca2 gene. The Brca2 deficiency in V-C8 cells causes hypersensitivity to various DNA-damaging agents with an extreme sensitivity toward interstrand DNA cross-linking agents.