Ogryzko, V. | bioRxiv Science

bioRxiv2024

PKA prevents resection of DNA double strand breaks and favor non-homologous end-joining

Genome rearrangement is a hallmark of cancer and ageing. DNA double-strand breaks (DSBs) are highly toxic lesions that can generate genome rearrangements. Several pathways compete for DSB repair, and selection of the appropriate repair process is decisive for genetic stability. DSB repair act according to two steps: 1-the choice between nonhomologous end-joining (NHEJ) versus resection of DNA ends, generating single-stranded DNA (ssDNA); 2-on ssDNA, homologous recombination (HR) and sub-processes. Here we show that 53BP1, which plays a prime role at the first step by protecting against resection and fostering NHEJ, physically interacts with the catalytic subunits of the cAMP dependant protein kinase A (PKAcs). PKA favors the recruitment of 53BP1 at DNA damages sites, and consistently, prevents resection, favoring NHEJ. Inhibition of PKA stimulated resection and coherently reduces NHEJ. Reversely, activation of PKA with 8-Bromo-cAMP stimulate NHEJ and reduces HR. These data open new avenues for potential anticancer strategies. More generally, they underline the high complexity of the regulation of DSB repair, identifying PKA as a novel actor of the DNA damage response, acting at the choice of the DSB repair process, which might be essential for genome integrity maintenance.

molecular biology↗

bioRxiv2018

Topokaryotyping demonstrates single cell variability and stress dependent variations in nuclear envelope associated domains

Analysis of large-scale interphase genome positioning with reference to a nuclear landmark has recently been studied using sequencing-based single cell approaches. However, these approaches are dependent upon technically challenging, time consuming and costly high throughput sequencing technologies, requiring specialized bioinformatics tools and expertise. Here, we propose a novel, affordable and robust microscopy-based single cell approach, termed Topokaryotyping, to analyze and reconstruct the interphase positioning of genomic loci relative to a given nuclear landmark, detectable as banding pattern on mitotic chromosomes. This is accomplished by proximity-dependent histone labeling, where biotin ligase BirA fused to nuclear envelope marker Emerin was coexpressed together with Biotin Acceptor Peptide (BAP)-histone fusion followed by (i) biotin labeling, (ii) generation of mitotic spreads, (iii) detection of the biotin label on mitotic chromosomes and (iv) their identification by karyotyping. Using Topokaryotyping, we identified both cooperativity and stochasticity in the positioning of emerin-associated chromatin domains in individual cells. Furthermore, the chromosome-banding pattern showed dynamic changes in emerin-associated domains upon physical and radiological stress. In summary, Topokaryotyping is a sensitive and reliable technique to quantitatively analyze spatial positioning of genomic regions interacting with a given nuclear landmark at the single cell level in various experimental conditions.

cell biology↗