John Thacker

Mutation in Brca2 stimulates error-prone homology-directed repair of DNA double-strand breaks occurring between repeated sequences.

Mutation of BRCA2 causes familial early onset breast and ovarian cancer. BRCA2 has been suggested to be important for the maintenance of genome integrity and to have a role in DNA repair by homology‐ directed double‐strand break (DSB) repair. By studying the repair of a specific induced chromosomal DSB we show that loss of Brca2 leads to a substantial increase in error‐prone repair by homology‐directed single‐strand annealing and a reduction in DSB repair by conservative gene conversion. These data demonstrate that loss of Brca2 causes misrepair of chromosomal DSBs occurring between repeated sequences by stimulating use of an error‐prone homologous recombination pathway. Furthermore, loss of Brca2 causes a large increase in genome‐wide error‐prone repair of both spontaneous DNA damage and mitomycin C‐induced DNA cross‐links at the expense of error‐free repair by sister chromatid recombination. This provides insight into the mechanisms that induce genome instability in tumour cells lacking BRCA2.

Mutation and inactivation of cultured mammalian cells exposed to beams of accelerated heavy ions. II. Chinese hamster V79 cells.

Inactivation and mutation to thioguanine-resistance of V79 hamster cells were studied after irradiation with accelerated helium, boron or nitrogen ions covering a range of linear energy transfer from 28 to 470 keV micrometers-1. For all radiation qualities a dose-dependent increase in mutant frequency was found for doses giving surviving fractions greater than about 0.20. The effectiveness per unit dose for both inactivation and mutation induction increased with the linear energy transfer of the radiation to a maximum in the range 90-200 keV micrometer-1. However, the maximum mutagenic effectiveness relative to gamma-rays was about two or more times that for inactivation. It is suggested that a proportion of the radiation-induced mutants suffer extensive genetic damage, and that some forms of this damage may be induced with high efficiency by radiations of high linear energy transfer.

Mammalian recombination-repair genes XRCC2 and XRCC3 promote correct chromosome segregation

Growth and development are dependent on the faithful duplication of cells. Duplication requires accurate genome replication, the repair of any DNA damage, and the precise segregation of chromosomes at mitosis; molecular checkpoints ensure the proper progression and fidelity of each stage. Loss of any of these highly conserved functions may result in genetic instability and proneness to cancer. Here we show that highly significant increases in chromosome missegregation occur in cell lines lacking the RAD51-like genes XRCC2 and XRCC3. This increased missegregation is associated with fragmentation of the centrosome, a component of the mitotic spindle, and not with loss of the spindle checkpoint. Our results show that unresolved DNA damage triggers this instability, and that XRCC2 and XRCC3 are potential tumour-suppressor genes in mammals.

Xrcc2 is required for genetic stability, embryonic neurogenesis and viability in mice

Repair of DNA damage by homologous recombination has only recently been established as an important mechanism in maintaining genetic stability in mammalian cells. The recently cloned Xrcc2 gene is a member of the mammalian Rad51 gene family, thought to be central to homologous recombination repair. To understand its function in mammals, we have disrupted Xrcc2 in mice. No Xrcc2−/− animals were found alive, with embryonic lethality occurring from mid‐gestation. Xrcc2−/− embryos surviving until later stages of embryogenesis commonly showed developmental abnormalities and died at birth. Neonatal lethality, apparently due to respiratory failure, was associated with a high frequency of apoptotic death of post‐ mitotic neurons in the developing brain, leading to abnormal cortical structure. Embryonic cells showed genetic instability, revealed by a high level of chromosomal aberrations, and were sensitive to γ‐rays. Our findings demonstrate that homologous recombination has an important role in endogenous damage repair in the developing embryo. Xrcc2 disruption identifies a range of defects that arise from malfunction of this repair pathway, and establishes a previously unidentified role for homologous recombination repair in correct neuronal development.

The nature of mutants induced by ionising radiation in cultured hamster cells III. Molecular characterization of HPRT-deficient mutants induced by γ-rays or α-particles showing that the majority have deletions of all or part of the hprt gene

DNA from 58 independent HPRT-deficient mutants of V79 hamster cells induced by ionising radiation was analysed by Southern blot hybridization to a full-length hamster hprt cDNA. About half of the gamma-ray-induced mutants (20/43) were apparently total gene deletions, because they lacked all functional hprt gene sequences hybridizing to the cDNA probe. Another 10 mutants showed various partial deletions and/or rearrangements of the hprt gene. The remaining 13 mutants showed no detectable change in comparison to the structure of the normal gene, which correlated well with previous characterization of these mutants indicating that most carry point mutations in the hprt gene. However, it is probable that some of these point mutations occurred spontaneously rather than being radiation-induced. A smaller number of alpha-particle induced mutants gave similar results: out of a total of 15 mutants, 6 appeared to be total gene deletions, 5 had partial deletions and/or rearrangements, and 4 had no detectable changes. Thus, 70% or more of radiation-induced HPRT-deficient mutants arise through large genetic changes, especially deletions of all or part of the hprt gene. This result is to be contrasted with data published previously by ourselves and others indicating that the majority of spontaneous and ethyl methanesulphonate-induced mutations of hprt and similar genes arise by point mutation.

Isolation and cross-sensitivity of X-ray-sensitive mutants of V79-4 hamster cells

The V79-4 Chinese hamster line was mutagenized and surviving clones screened for X-ray sensitivity using a replica microwell technique. One slightly sensitive clone and 3 clearly sensitive clones were isolated from approximately 5000 screened, and designated irs 1 to irs 4. The 3 more sensitive clones showed different responses to the genotoxic agents mitomycin C (MMC), ethyl methanesulphonate (EMS) and ultraviolet light (UV). irs 1 showed considerable sensitivity to all the agents tested, in the order MMC much greater than EMS greater than UV. irs 2 and irs 3 had similar sensitivities to EMS and to UV (EMS greater than UV) but irs 3 was more sensitive than irs 2 to MMC. None of these mutants is identical in phenotype to previously published mutants.

A surfeit of RAD51-like genes?

The process of homologous recombination appears enigmatic: it creates genetic diversity but also provides an important way of repairing DNA damage without errors. The recent cloning and functional analysis of eukaryotic recombination genes suggests that at least part of the intricate mechanism involved is conserved from bacteria to humans, but is also yielding some surprises.

The XRCC2 DNA repair gene from human and mouse encodes a novel member of the recA/RAD51 family

We recently identified a positional candidate for the XRCC2 DNA repair gene at human chromosome 7q36.1. We have now cloned the cDNA for this gene from both human and mouse and show that it is a highly conserved novel member of the recA / RAD51 recombination repair gene family. The cDNA is able to complement significantly the phenotype of a unique cell line, irs1 , which shows extreme sensitivity to DNA cross-linking agents and genetic instability. Thisphenotype is consistent with a role for the XRCC2 gene in recombination repair in somatic cells, suggesting that in addition to RAD51 , other members of this gene family have an important function in high fidelity repair processes in mammals. Despite this function, the XRCC2 gene transcript is expressed at a very low level in somatic tissue, but is elevated in mouse testis, suggesting an additional role in meiosis.

Inactivation and mutation of cultured mammalian cells by aluminium characteristic ultrasoft X-rays I. Properties of aluminium X-rays and preliminary experiments with Chinese hamster cells

Irradiation with ultrasoft X-rays produces electron tracks of short defined lengths in the irradiated material. This property is of particular interest in distinguishing between different models of radiation action on living organisms. The production, absorption and dosimetry of aluminium K characteristic X-rays of energy 1.5 keV are described. Quantitative experiments on mammalian cells with these X-rays are possible, and they were found to be considerably more effective than gamma-rays in inactivating Chinese hamster V79 cells in vitro.

The mammalian XRCC genes: their roles in DNA repair and genetic stability.

Analysis of the XRCC genes has played an important part in understanding mammalian DNA repair processes, especially those involved in double-strand break (DSB) repair. Most of these genes were identified through their ability to correct DNA damage hypersensitivity in rodent cell lines, and they represent components of several different repair pathways including base-excision repair, non-homologous end joining, and homologous recombination. We document the phenotypic effects of mutation of the XRCC genes, and the current state of our knowledge of their functions. In addition to their continuing importance in discovering mechanisms of DNA repair, analysis of the XRCC genes is making a substantial contribution to the understanding of specific human disorders, including cancer.