BRCA1 recruitment to damaged DNA sites is dependent on CDK9

<p>Double strand break lesions, the most toxic type of DNA damage, are repaired primarily through 2 distinct pathways: homology-directed recombination (HR) and non-homologous end-joining (NHEJ). BRCA1 and 53BP1, 2 proteins containing the BRCT modular domain, play an important role in DNA damage response (DDR) by orchestrating the decision between HR and NHEJ, but the precise mechanisms regarding both pathways are not entirely understood. Previously, our group identified a putative interaction between BRCA1 and BARD1 (BRCA1-associated RING domain 1) and the cyclin-dependent kinase (CDK9). CDK9 is a component of the positive transcription elongation complex and has been implicated in genome integrity maintenance associated with the replication stress response. Here we show that CDK9 interacts with endogenous BRCA1 and BARD1 mediated by their RING finger and BRCT domains, and describe CDK9 ionizing radiation-induced foci (IRIF) formation and its co-localization with BRCA1 in DNA damage sites. Cells lacking CDK9 are characterized by an altered γ−H2AX foci dynamics after DNA damage, a reduced efficiency in HR but not in NHEJ repair, failure to form BRCA1 and RAD51 IRIF and increased sensitivity to genotoxic agents. These data indicate that CDK9 is a player in the DDR and is consistent with its participation in HR pathway by modulating BRCA1 response.</p

Probing Structure-Function Relationships in Missense Variants in the Carboxy-Terminal Region of BRCA1

<div><p>Germline inactivating variants in <i>BRCA1</i> lead to a significantly increased risk of breast and ovarian cancers in carriers. While the functional effect of many variants can be inferred from the DNA sequence, determining the effect of missense variants present a significant challenge. A series of biochemical and cell biological assays have been successfully used to explore the impact of these variants on the function of BRCA1, which contribute to assessing their likelihood of pathogenicity. It has been determined that variants that co-localize with structural or functional motifs are more likely to disrupt the stability and function of BRCA1. Here we assess the functional impact of 37 variants chosen to probe the functional impact of variants in phosphorylation sites and in the BRCT domains. In addition, we perform a meta-analysis of 170 unique variants tested by the transcription activation assays in the carboxy-terminal domain of BRCA1 using a recently developed computation model to provide assessment for functional impact and their likelihood of pathogenicity.</p></div

Functional analysis of missense variants in BRCA1 C-terminal region.

<p>Transcriptional activity of BRCA1 variants were evaluated in HEK293T cells using a GAL4-responsive firefly luciferase reporter gene (shown above the graphs) at 37°C. Cells were harvested 24h after transfections and the lysate was used to assess transcriptional activation ability by luciferase activity measurement. Activity is depicted as % of the wild-type activity. <i>(</i><b><i>A</i></b><i>)</i> Natural missense variants and <i>(</i><b><i>B</i></b><i>)</i> natural and artificial (underlined) variants located on phosphorylation sites. S1613G, M1775R and Y1853X variants were used as controls.</p

Estimated Variant Specific Effects (Bayesian statistical model, VarCall, graphical summary).

<p>Variants are depicted in order of amino acid from residues 1396 to 1863. The top panel shows secondary structures in the C-terminal region. Coiled-coil region and α-helixes are depicted as pink and blue boxes, respectively. β-sheets are shown as gray arrows. The shaded area on the graph corresponds to different structures of similar color. The linker region is indicated with green shading. Each variant’s activity is represented by a boxplot summarizing the marginal posterior distribution of its random effect. A point estimate of the mixture model is plotted on the right margin. Its top component corresponds to variants with no functional impact, whereas its bottom component corresponds to variants with functional impact. The mean of the benign/damaging component is plotted as a green/red dotted horizontal line. Yellow box represents wild-type reference, green and red boxes represent low (class 1 and 2) and high (class 4 and 5) risk variants respectively, classified according to the International Agency for Research on Cancer (IARC), purple boxes represent VUS previously analyzed by our group (used to feed VarClass algorithm), and blue boxes represent the VUS analyzed by the first time in this study.</p