Synthesis-Dependent Strand Annealing in Meiosis

Recent studies led to the proposal that meiotic gene conversion can result after transient engagement of the donor chromatid and subsequent DNA synthesis-dependent strand annealing (SDSA). Double Holliday junction (dHJ) intermediates were previously proposed to form both reciprocal crossover recombinants (COs) and noncrossover recombinants (NCOs); however, dHJs are now thought to give rise mainly to COs, with SDSA forming most or all NCOs. To test this model in Saccharomyces cerevisiae, we constructed a random spore system in which it is possible to identify a subset of NCO recombinants that can readily be accounted for by SDSA, but not by dHJ-mediated recombination. The diagnostic class of recombinants is one in which two markers on opposite sides of a double-strand break site are converted, without conversion of an intervening heterologous insertion located on the donor chromatid. This diagnostic class represents 26% of selected NCO recombinants. Tetrad analysis using the same markers provided additional evidence that SDSA is a major pathway for NCO gene conversion in meiosis

Non-enzymatic roles of human RAD51 at stalled replication forks

The central recombination enzyme RAD51 has been implicated in replication fork processing and restart in response to replication stress. Here, we use a separation-of-function allele of RAD51 that retains DNA binding, but not D-loop activity, to reveal mechanistic aspects of RAD51’s roles in the response to replication stress. Here, we find that cells lacking RAD51’s enzymatic activity protect replication forks from MRE11-dependent degradation, as expected from previous studies. Unexpectedly, we find that RAD51’s strand exchange activity is not required to convert stalled forks to a form that can be degraded by DNA2. Such conversion was shown previously to require replication fork regression, supporting a model in which fork regression depends on a non-enzymatic function of RAD51. We also show RAD51 promotes replication restart by both strand exchange-dependent and strand exchange-independent mechanisms

RAD54 family translocases counter genotoxic effects of RAD51 in human tumor cells.

The RAD54 family DNA translocases have several biochemical activities. One activity, demonstrated previously for the budding yeast translocases, is ATPase-dependent disruption of RAD51-dsDNA binding. This activity is thought to promote dissociation of RAD51 from heteroduplex DNA following strand exchange during homologous recombination. In addition, previous experiments in budding yeast have shown that the same activity of Rad54 removes Rad51 from undamaged sites on chromosomes; mutants lacking Rad54 accumulate nonrepair-associated complexes that can block growth and lead to chromosome loss. Here, we show that human RAD54 also promotes the dissociation of RAD51 from dsDNA and not ssDNA. We also show that translocase depletion in tumor cell lines leads to the accumulation of RAD51 on chromosomes, forming complexes that are not associated with markers of DNA damage. We further show that combined depletion of RAD54L and RAD54B and/or artificial induction of RAD51 overexpression blocks replication and promotes chromosome segregation defects. These results support a model in which RAD54L and RAD54B counteract genome-destabilizing effects of direct binding of RAD51 to dsDNA in human tumor cells. Thus, in addition to having genome-stabilizing DNA repair activity, human RAD51 has genome-destabilizing activity when expressed at high levels, as is the case in many human tumors

A comparative analysis of Dmc1 and Rad51 nucleoprotein filaments

filament

The Epistatic Relationship between BRCA2 and the Other RAD51 Mediators in Homologous Recombination

RAD51 recombinase polymerizes at the site of double-strand breaks (DSBs) where it performs DSB repair. The loss of RAD51 causes extensive chromosomal breaks, leading to apoptosis. The polymerization of RAD51 is regulated by a number of RAD51 mediators, such as BRCA1, BRCA2, RAD52, SFR1, SWS1, and the five RAD51 paralogs, including XRCC3. We here show that brca2-null mutant cells were able to proliferate, indicating that RAD51 can perform DSB repair in the absence of BRCA2. We disrupted the BRCA1, RAD52, SFR1, SWS1, and XRCC3 genes in the brca2-null cells. All the resulting double-mutant cells displayed a phenotype that was very similar to that of the brca2-null cells. We suggest that BRCA2 might thus serve as a platform to recruit various RAD51 mediators at the appropriate position at the DNA–damage site.</p

Scattering statistics of rock outcrops: Model-data comparisons and Bayesian inference using mixture distributions

The probability density function of the acoustic field amplitude scattered by the seafloor was measured in a rocky environment off the coast of Norway using a synthetic aperture sonar system, and is reported here in terms of the probability of false alarm. Interpretation of the measurements focused on finding appropriate class of statistical models (single versus two-component mixture models), and on appropriate models within these two classes. It was found that two-component mixture models performed better than single models. The two mixture models that performed the best (and had a basis in the physics of scattering) were a mixture between two K distributions, and a mixture between a Rayleigh and generalized Pareto distribution. Bayes' theorem was used to estimate the probability density function of the mixture model parameters. It was found that the K-K mixture exhibits significant correlation between its parameters. The mixture between the Rayleigh and generalized Pareto distributions also had significant parameter correlation, but also contained multiple modes. We conclude that the mixture between two K distributions is the most applicable to this dataset.Comment: 15 pages, 7 figures, Accepted to the Journal of the Acoustical Society of Americ

The Breast Cancer Susceptibility Gene BRCA1 Is Required for Subnuclear Assembly of Rad51 and Survival following Treatment with the DNA Cross-linking Agent Cisplatin

Mutations in breast cancer tumor susceptibility genes, BRCA1 and BRCA2, predispose women to early onset breast cancer and other malignancies. The Brca genes are involved in multiple cellular processes in response to DNA damage including checkpoint activation, gene transcription, and DNA repair. Biochemical interaction with the recombinational repair protein Rad51 (Scully, R., Chen, J., Ochs, R. L., Keegan, K., Hoekstra, M., Feunteun, J., and Livingston, D. M. (1997) Cell 90, 425-435), as well as genetic evidence (Moynahan, M. E., Chiu, J. W., Koller, B. H., and Jasin, M. (1999) Mol. Cell 4, 511-518 and Snouwaert, J. N., Gowen, L. C., Latour, A. M., Mohn, A. R., Xiao, A., DiBiase, L., and Koller, B. H. (1999) Oncogene 18, 7900-7907), demonstrates that Brca1 is involved in recombinational repair of DNA double strand breaks. Using isogenic Brca1(+/+) and brca1(-/-) mouse embryonic stem (ES) cell lines, we investigated the role of Brca1 in the cellular response to two different categories of DNA damage: x-ray induced damage and cross-linking damage caused by the chemotherapeutic agent, cisplatinum. Immunoflourescence studies with normal and brca1(-/-) mutant mouse ES cell lines indicate that Brca1 promotes assembly of subnuclear Rad51 foci following both types of DNA damage. These foci are likely to be oligomeric complexes of Rad51 engaged in repair of DNA lesions or in processes that allow cells to tolerate such lesions during DNA replication. Clonogenic assays show that brca1(-/-) mutants are 5-fold more sensitive to cisplatinum compared with wild-type cells. Our studies suggest that Brca1 contributes to damage repair and/or tolerance by promoting assembly of Rad51. This function appears to be shared with Brca2

The Reframing of Methodology: Revisiting a PhD Study

The paper draws on a PhD study to explore some methodological dilemmas associated with the execution of qualitative research when framed within positivist study design. The PhD was linked to an externally funded research project which evaluated the implementation of a custody-based intervention in the secure estate. While the PhD was conceived as a qualitative study, informed by interpretivist methodology and associated epistemology, the wider funded study was informed by positivist tradition and used a quantitative method. This led to dilemmas of both practical and methodological nature. The author revisits her study's methodological position to review issues raised by the research design and suggests an alternative proposal informed methodologically by critical realism which may better serve the study's interests. In doing so, the paper suggests how revisiting previous research may assist us in gaining methodological understanding and allow us to reframe our future endeavours to more useful end