The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein. We developed a system whereby the endogenous BRCA1 pr...

Gene targeting is a challenge in organisms where non-homologous end-joining is the predominant form of recombination. We show that cell division cycle synchronization can be applied to significantly increase the rate of homologous recombination during transformation. Using hydroxyurea-mediated cell cycle arrest, we obtained improved gene targeting rates in Yarrowia lipolytica, Arxula adeninivor...

Chromosomal double-strand DNA breaks must be repaired; in the absence of repair the resulting acentromeric (and telomereless) fragments may be lost and/or the broken DNA ends may recombine causing general chromosomal instability. The Rad50/Mre11/Xrs2 protein complex acts at DNA ends and is implicated in both homologous and non-homologous recombination. We have isolated a rad50 mutant of the pla...

Homologous recombination is a universal mechanism that allows repair of DNA and provides support for DNA replication. Homologous recombination is therefore a major pathway that suppresses non-homology-mediated genome instability. Here, we report that recovery of impeded replication forks by homologous recombination is error-prone. Using a fork-arrest-based assay in fission yeast, we demonstrate...

The process of homologous recombination is indispensable for both meiotic and mitotic cell division, and is one of the major pathways for double-strand break (DSB) repair. The human Rad54B protein, which belongs to the SWI2/SNF2 protein family, plays a role in homologous recombination, and may function with the Dmc1 recombinase, a meiosis-specific Rad51 homolog. In the present study, we found t...

Double-strand DNA break (DSB) repair by homologous recombination occurs through the RAD52 pathway in Saccharomyces cerevisiae. Its biological importance is underscored by the conservation of many RAD52 pathway genes, including RAD54, from fungi to humans. We have analyzed the phenotype of mouse RAD54-/- (mRAD54-/-) cells. Consistent with a DSB repair defect, these cells are sensitive to ionizin...

We report a PCR-induced artifact in testing for homologous recombination in zebrafish. We attempted to replace the lnx2a gene with a donor cassette, mediated by a TALEN induced double stranded cut. The donor construct was flanked with homology arms of about 1 kb at the 5' and 3' ends. Injected embryos (G0) were raised and outcrossed to wild type fish. A fraction of the progeny appeared to have ...

Pairing, synapsis, and recombination are prerequisites for accurate chromosome segregation in meiosis. The phs1 gene in maize is required for pairing to occur between homologous chromosomes. In the phs1 mutant, homologous chromosome synapsis is completely replaced by synapsis between nonhomologous partners. The phs1 gene is also required for installation of the meiotic recombination machinery o...

We have developed a versatile plasmid vector (pReco-sigma) for recombination studies. When linearized and introduced into the cells of interest, pReco-sigma allows the simultaneous determination of the relative frequencies of homologous recombination versus nonhomologous DNA-end joining (also termed end-to-end joining), the latter an example of illegitimate recombination processes. As a system ...