Truncated BRCA2 is cytoplasmic: implications for cancer-linked mutations.

BRCA2 mutations predispose carriers mainly to breast cancer. The vast majority of BRCA2 mutations are predicted to result in a truncated protein product. The smallest known cancer-associated deletion removes from the C terminus only 224 of the 3,418 residues constituting BRCA2, suggesting that these terminal amino acids are crucial for BRCA2 function. A series of green fluorescent protein (GFP)-tagged BRCA2 deletion mutants revealed that nuclear localization depends on two nuclear localization signals that reside within the final 156 residues of BRCA2. Consistent with this observation, an endogenous truncated BRCA2 mutant (6174delT) was found to be cytoplasmic. Together, these studies provide a simple explanation for why the vast majority of BRCA2 mutants are nonfunctional: they do not translocate into the nucleus.

Two human cDNAs, including a homolog of Arabidopsis FUS6 (COP11), suppress G-protein- and mitogen-activated protein kinase-mediated signal transduction in yeast and mammalian cells.

We have isolated two novel human cDNAs, gps1-1 and gps2, that suppress lethal G-protein subunit-activating mutations in the pheromone response pathway of the yeast Saccharomyces cerevisiae. Suppression of other pathway-activating events was examined. In wild-type cells, expression of either gps1-1 or gps2 led to enhanced recovery from cell cycle arrest induced by pheromone. Sequence analysis indicated that gps1-1 contains only the carboxy-terminal half of the gps1 coding sequence. The predicted gene product of gps1 has striking similarity to the protein encoded by the Arabidopsis FUS6 (COP11) gene, a negative regulator of light-mediated signal transduction that is known to be essential for normal development. A chimeric construct containing gps1 and FUS6 sequences also suppressed the yeast pheromone pathway, indicating functional conservation between these human and plant genes. In addition, when overexpressed in mammalian cells, gps1 or gps2 potently suppressed a RAS- and mitogen-activated protein kinase-mediated signal and interfered with JNK activity, suggesting that signal repression is part of their normal function. For gps1, these results are consistent with the proposed function of FUS6 (COP11) as a signal transduction repressor in plants.

Truncated forms of a novel yeast protein suppress the lethality of a G protein alpha subunit deficiency by interacting with the beta subunit.

In Saccharomyces cerevisiae, the mating pheromone-initiated signal is transduced by a heterotrimeric G protein and normally results in transient cell cycle arrest and differentiation. A null allele of the G alpha (GPA1/SCG1) subunit results in cell death due to unchecked signaling from the G beta gamma (STE4, STE18, respectively) heterodimer. We have identified three high copy suppressors of gpa1 lethality. Two of these genes encode known transcription factors. Mat alpha 2p and Mcm1p. The third is a truncated form of a novel gene, SYG1. Overexpressed wild type SYG1 is a weak suppressor of gpa1. In contrast, the isolated mutant allele SYG1-1 is a strong suppressor that completely blocks the cell cycle arrest and differentiation phenotypes of gpa1 cells of both mating types. One deletion mutant (SYG1 delta 340) can suppress the cell cycle arrest associated with gpa1, but the cells retain a differentiated morphology. SYG1-1 can suppress the effects of overexpressed wild type G beta but is not able to suppress the lethality of an activated G beta mutant (STE4Hpl). Consistent with these genetic observations, the suppressing form of Syg1p can interact with the STE4 gene product, as determined by a two-hybrid assay. SYG1-1 is also capable of promoting pheromone recovery in wild type cells, as judged by halo assay. The sequence of SYG1 predicts eight membrane-spanning domains. Deletion mutants of SYG1 indicate that complete gpa1 suppression requires removal of all of these hydrophobic regions. Interestingly, this truncated protein localizes to the same plasma membrane-enriched subcellular fraction as does full-length Syg1p. Three hypothetical yeast proteins, identified by their similarity to the SYG1 primary sequence within the gpa1 suppression domain, also appear to have related structures. The properties of Syg1p are consistent with those of a transmembrane signaling component that can respond to, or transduce signals through, G beta or G beta gamma.

Gene expression of human DNA polymerase alpha during cell proliferation and the cell cycle.

We studied the expression of the human DNA polymerase alpha gene during cell proliferation, during cell progression through the cell cycle, and in transformed cells compared with normal cells. During the activation of quiescent cells (G0 phase) to proliferate (G1/S phases), the steady-state mRNA levels, rate of synthesis of nascent polymerase protein, and enzymatic activity in vitro exhibited a substantial and concordant increase prior to the peak of in vivo DNA synthesis. In transformed cells, the respective values were amplified greater than 10-fold. In actively growing cells separated into discrete stages of the cell cycle by counterflow elutriation or by mitotic shakeoff, levels of steady-state transcripts, translation rates, and enzymatic activities of polymerase alpha were constitutively and concordantly expressed at all stages of the cell cycle, with only a moderate elevation prior to the S phase and a slight decline in the G2 phase. These findings support the conclusion that the regulation of human DNA polymerase alpha gene expression is at the transcriptional level and strongly suggest that the regulatory mechanisms that are operative during the entrance of a cell into the mitotic cycle are fundamentally different from those that modulate polymerase alpha expression in continuously cycling cells.