DNA polymerase zeta contributes to heterochromatin replication to prevent genome instability.

The DNA polymerase zeta (Polζ) plays a critical role in bypassing DNA damage. REV3L, the catalytic subunit of Polζ, is also essential in mouse embryonic development and cell proliferation for reasons that remain incompletely understood. In this study, we reveal that REV3L protein interacts with heterochromatin components including repressive histone marks and localizes in pericentromeric regions through direct interaction with HP1 dimer. We demonstrate that Polζ/REV3L ensures progression of replication forks through difficult-to-replicate pericentromeric heterochromatin, thereby preventing spontaneous chromosome break formation. We also find that Rev3l-deficient cells are compromised in the repair of heterochromatin-associated double-stranded breaks, eliciting deletions in late-replicating regions. Lack of REV3L leads to further consequences that may be ascribed to heterochromatin replication and repair-associated functions of Polζ, with a disruption of the temporal replication program at specific loci. This is correlated with changes in epigenetic landscape and transcriptional control of developmentally regulated genes. These results reveal a new function of Polζ in preventing chromosome instability during replication of heterochromatic regions.

Functional Assessment of Genetic Variants with Outcomes Adapted to Clinical Decision-Making.

Understanding the medical effect of an ever-growing number of human variants detected is a long term challenge in genetic counseling. Functional assays, based on in vitro or in vivo evaluations of the variant effects, provide essential information, but they require robust statistical validation, as well as adapted outputs, to be implemented in the clinical decision-making process. Here, we assessed 25 pathogenic and 15 neutral missense variants of the BRCA1 breast/ovarian cancer susceptibility gene in four BRCA1 functional assays. Next, we developed a novel approach that refines the variant ranking in these functional assays. Lastly, we developed a computational system that provides a probabilistic classification of variants, adapted to clinical interpretation. Using this system, the best functional assay exhibits a variant classification accuracy estimated at 93%. Additional theoretical simulations highlight the benefit of this ready-to-use system in the classification of variants after functional assessment, which should facilitate the consideration of functional evidences in the decision-making process after genetic testing. Finally, we demonstrate the versatility of the system with the classification of siRNAs tested for human cell growth inhibition in high throughput screening.

Yeast cells reveal the misfolding and the cellular mislocalization of the human BRCA1 protein.

Understanding the effect of an ever-growing number of human variants detected by genome sequencing is a medical challenge. The yeast Saccharomyces cerevisiae model has held attention for its capacity to monitor the functional impact of missense mutations found in human genes, including the BRCA1 breast and ovarian cancer susceptibility gene. When expressed in yeast, the wild-type full-length BRCA1 protein forms a single nuclear aggregate and induces a growth inhibition. Both events are modified by pathogenic mutations of BRCA1. However, the biological processes behind these events in yeast remain to be determined. Here, we show that the BRCA1 nuclear aggregation and the growth inhibition are sensitive to misfolding effects induced by missense mutations. Moreover, misfolding mutations impair the nuclear targeting of BRCA1 in yeast cells and in a human cell line. In conclusion, we establish a connection between misfolding and nuclear transport impairment, and we illustrate that yeast is a suitable model to decipher the effect of misfolding mutations.