Fonction et régulation de l'ADN polymérase zêta au cours de la réplication de l'ADN : conséquences sur la stabilité du génome.

DNA replication is a fundamental process that ensures accurate duplication of the genetic information. Various perturbations can impede replication fork progression, and thus threatening genome integrity. To prevent fork collapse, replicative DNA polymerases can be replaced by error-prone DNA polymerases called translesion (TLS) polymerases, able to bypass DNA damage at the cost of increased mutations. Among TLS polymerases, Polζ is unique because inactivation of its catalytic subunit, REV3L, leads to embryonic lethality in mice underscoring its biological importance. However, little is known about its function and regulation in mammalian cells. We showed that loss of REV3L impairs S phase progression with a disruption of replication timing at specific genomic loci that replicate in mid-late S-phase, and this is associated with increased mutagenic events and aberrant epigenetic landscape. We also revealed that REV3L interacts with heterochromatin components and localizes in pericentromeric regions, suggesting that Polζ contributes to replicate heterochromatin regions to limit genome instability. In a second part, we discovered that REV3L protein is proteolytically processed by the endopeptidase TASP1 to generate two polypeptides that heterodimerize to form a stable complex that associates with REV7, likely representing the active complex of Polζ. We also found that REV3L is finely regulated in physiological conditions and after genotoxic stress at multiple levels: (1) transcriptionally, (2) proteolytically by TASP1 and (3) post-translationally by phosphorylation. Altogether these findings highlight a unique mechanism to control the function of an error-prone polymerase in mammalian cells. These data are particularly important given that Polζ is an important factor for tumor resistance to chemotherapeutic agents.La réplication de l’ADN est un processus cellulaire fondamental qui assure la duplication fidèle de l’information génétique. Différentes perturbations peuvent interférer avec la progression de la fourche de réplication menaçant ainsi l’intégrité du génome. Pour éviter le blocage du réplisome, les polymérases réplicatives peuvent être remplacées par des polymérases translésionnelles (TLS) mutagènes mais capables de franchir différents types de lésions. Parmi les polymérases TLS, Polζ est unique car son absence entraine une létalité embryonnaire chez la souris suggérant qu’elle a acquis une fonction essentielle au cours de l’évolution. Cependant, sa fonction et sa régulation dans les cellules mammifères restent méconnues. Dans ce travail, nous avons montré que la phase S est perturbée en absence de REV3L, avec une modification du programme temporel de la réplication au niveau de régions génomiques répliquées en milieu-fin de phase S. Ce défaut de réplication est associé à une augmentation de la mutagénèse et des modifications du paysage épigénétique. Nous avons de plus mis en évidence que REV3L interagit avec les composants de l’hétérochromatine et est localisé au niveau des régions péri-centromériques, ce qui suggère que Polζ participe à la réplication de l’hétérochromatine et limite ainsi l’instabilité génomique. Dans une seconde partie, nous avons découvert que la protéine REV3L est clivée de manière post-traductionnelle par l’endopeptidase TASP1 générant deux polypeptides capables de s’hétérodimériser pour former un complexe stable qui, en association avec Rev7, représente probablement le complexe actif de Polζ. Aussi, nous avons observé que REV3L est finement régulée de manière endogène ou après exposition à un stress génotoxique à de multiples niveaux : (1) au niveau transcriptionnel, (2) par le clivage par TASP1, (3) par des phosphorylations post-traductionnelles. Finalement, l’ensemble de ces découvertes met en lumière un mécanisme de régulation unique contrôlant la fonction d’une polymérase mutagène dans les cellules mammifères. Ces résultats sont particulièrement importants étant donné que Polζ est un facteur impliqué dans les mécanismes de résistance des tumeurs face aux chimiothérapies

Function and Regulation of the DNA Polymerase Zeta during the DNA Replication : Consequences on Genome Stability.

La réplication de l’ADN est un processus cellulaire fondamental qui assure la duplication fidèle de l’information génétique. Différentes perturbations peuvent interférer avec la progression de la fourche de réplication menaçant ainsi l’intégrité du génome. Pour éviter le blocage du réplisome, les polymérases réplicatives peuvent être remplacées par des polymérases translésionnelles (TLS) mutagènes mais capables de franchir différents types de lésions. Parmi les polymérases TLS, Polζ est unique car son absence entraine une létalité embryonnaire chez la souris suggérant qu’elle a acquis une fonction essentielle au cours de l’évolution. Cependant, sa fonction et sa régulation dans les cellules mammifères restent méconnues. Dans ce travail, nous avons montré que la phase S est perturbée en absence de REV3L, avec une modification du programme temporel de la réplication au niveau de régions génomiques répliquées en milieu-fin de phase S. Ce défaut de réplication est associé à une augmentation de la mutagénèse et des modifications du paysage épigénétique. Nous avons de plus mis en évidence que REV3L interagit avec les composants de l’hétérochromatine et est localisé au niveau des régions péri-centromériques, ce qui suggère que Polζ participe à la réplication de l’hétérochromatine et limite ainsi l’instabilité génomique. Dans une seconde partie, nous avons découvert que la protéine REV3L est clivée de manière post-traductionnelle par l’endopeptidase TASP1 générant deux polypeptides capables de s’hétérodimériser pour former un complexe stable qui, en association avec Rev7, représente probablement le complexe actif de Polζ. Aussi, nous avons observé que REV3L est finement régulée de manière endogène ou après exposition à un stress génotoxique à de multiples niveaux : (1) au niveau transcriptionnel, (2) par le clivage par TASP1, (3) par des phosphorylations post-traductionnelles. Finalement, l’ensemble de ces découvertes met en lumière un mécanisme de régulation unique contrôlant la fonction d’une polymérase mutagène dans les cellules mammifères. Ces résultats sont particulièrement importants étant donné que Polζ est un facteur impliqué dans les mécanismes de résistance des tumeurs face aux chimiothérapies.DNA replication is a fundamental process that ensures accurate duplication of the genetic information. Various perturbations can impede replication fork progression, and thus threatening genome integrity. To prevent fork collapse, replicative DNA polymerases can be replaced by error-prone DNA polymerases called translesion (TLS) polymerases, able to bypass DNA damage at the cost of increased mutations. Among TLS polymerases, Polζ is unique because inactivation of its catalytic subunit, REV3L, leads to embryonic lethality in mice underscoring its biological importance. However, little is known about its function and regulation in mammalian cells. We showed that loss of REV3L impairs S phase progression with a disruption of replication timing at specific genomic loci that replicate in mid-late S-phase, and this is associated with increased mutagenic events and aberrant epigenetic landscape. We also revealed that REV3L interacts with heterochromatin components and localizes in pericentromeric regions, suggesting that Polζ contributes to replicate heterochromatin regions to limit genome instability. In a second part, we discovered that REV3L protein is proteolytically processed by the endopeptidase TASP1 to generate two polypeptides that heterodimerize to form a stable complex that associates with REV7, likely representing the active complex of Polζ. We also found that REV3L is finely regulated in physiological conditions and after genotoxic stress at multiple levels: (1) transcriptionally, (2) proteolytically by TASP1 and (3) post-translationally by phosphorylation. Altogether these findings highlight a unique mechanism to control the function of an error-prone polymerase in mammalian cells. These data are particularly important given that Polζ is an important factor for tumor resistance to chemotherapeutic agents

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

DNA polymerase zeta contributes to heterochromatin replication to prevent genome instability

International audienceThe DNA polymerase zeta (Polf) plays a critical role in bypassing DNA damage. REV3L, the catalytic subunit of Polf, 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 Polf/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 Polf, 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 Polf in preventing chromosome instability during replication of heterochromatic regions

Relative position of the variants in the Colony Size assay and fluctuation of the best cut-off.

<p>(<b>A</b>) Waterfall distribution of colony sizes, according to median values (standard method). Boxplot representation results from 9 (mutants) or 36 (BRCA1 and Vector) colony size values. The red and blue colors of the boxes indicate the pathogenic and neutral mutations, respectively, according to their prior classification. Box central bar, median; box, interquartile range (50% of the distribution); whiskers, extreme values; dotted horizontal line, median of BRCA1; thick horizontal line, experimental best cut-off (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006096#pgen.1006096.s004" target="_blank">S2 Fig</a>). The distribution of the best cut-off fluctuation, obtained after random sampling (bootstrap), of the 9 mutants and 36 BRCA1 values, is visualized by the pink, grey and light blue areas, that delimit 4%, 90% and 4.9% of the distribution, respectively, which altogether represents a total coverage of 98.9%. (<b>B</b>) Waterfall distribution according to p values (MWW method). The p value assigned to each variant is symbolized by a segment. The upside-down representation facilitates the comparison of the mutation arrangement with the one obtained in <b>A</b>. Arrows pinpoint a modification of the mutation rank depending on the method used. Framed mutations indicate identical p values (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006096#pgen.1006096.s033" target="_blank">S4 Table</a>). Segment colors, thick horizontal line and colored areas, as in <b>A</b>.</p

Variant classification using the probability system.

<p>(<b>A</b>) Schematic of the probability system of classification. The left figure depicts a theoretical waterfall distribution of pathogenic and neutral missense mutations, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006096#pgen.1006096.g001" target="_blank">Fig 1B</a>. Horizontal black line, experimental best cut-off. (1) Variant classification according to the experimental best cut-off (method used in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006096#pgen.1006096.t001" target="_blank">Table 1</a>). (2) Distribution of the best cut-off generated by bootstrap analysis from the experimental data. (3) Cumulative distribution function (CDF) derived from the distribution of the best cut-off. This CDF provides a probabilistic classification of the variants, depending on their positions in the CDF. (<b>B</b>) Classification of the <i>BRCA1</i> variants assessed in four functional assays. Colored background in the table indicates the five-class nomenclature, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006096#pgen.1006096.s030" target="_blank">S1 Table</a>. Names in red and blue indicate the pathogenic and neutral mutations, respectively, according to their prior classification. The sensitivity, specificity and accuracy computation are detailed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006096#pgen.1006096.s035" target="_blank">S6 Table</a>.</p

Experimental sensitivity, specificity and accuracy of functional assays and siRNA screening using the experimental best cut-off.

<p>Experimental sensitivity, specificity and accuracy of functional assays and siRNA screening using the experimental best cut-off.</p

High throughput siRNA screening and fluctuation of the best cut-off.

<p>An average of 150 human prostate tumoral cells were plated, treated with siRNAs targeting the indicated gene and grown for 72 hours before cell counting. The WT reference (No siRNA), the positive control of cell growth inhibition (siKIF11), the two negative controls of cell growth inhibition (siGOLGA2 and siGL2) and 8 among 406 siRNA targeted genes are shown. The complete analysis of the 406 targeted genes is available using the ProClass toolbox and the included siRNA full.txt file, as explained at the end of the README.doc file. (<b>A</b>) Waterfall distribution of cell growth after siRNA treatment, according to median values (standard method). As in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006096#pgen.1006096.g001" target="_blank">Fig 1A</a>, except that boxplot representation results from 12 (siRNA) or 1,140 (No siRNA) values. (<b>B</b>) Waterfall distribution according to p values (MWW method), as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006096#pgen.1006096.g001" target="_blank">Fig 1B</a>. (<b>C</b>) Classification of the siRNA targeted genes, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006096#pgen.1006096.g002" target="_blank">Fig 2B</a>, except that probabilities are related to cell growth inhibition, with the corresponding five-class nomenclature: "no inhibition" (blue, class1), "likely no inhibition" (light blue, class2), "unclear inhibition" (grey, class3), "likely inhibition" (light red, class4) and "inhibition" (red, class5).</p