Increased Gene Targeting in Hyper-Recombinogenic LymphoBlastoid Cell Lines Leaves Unchanged DSB Processing by Homologous Recombination

Abstract

In the cells of higher eukaryotes, sophisticated mechanisms have evolved to repair DNA double-strand breaks (DSBs). Classical nonhomologous end joining (c-NHEJ), homologous recombination (HR), alternative end joining (alt-EJ) and single-strand annealing (SSA) exploit distinct principles to repair DSBs throughout the cell cycle, resulting in repair outcomes of different fidelity. In addition to their functions in DSB repair, the same repair pathways determine how cells integrate foreign DNA or rearrange their genetic information. As a consequence, random integration of DNA fragments is dominant in somatic cells of higher eukaryotes and suppresses integration events at homologous genomic locations, leading to very low gene-targeting efficiencies. However, this response is not universal, and embryonic stem cells display increased targeting efficiency. Additionally, lymphoblastic chicken and human cell lines DT40 and NALM6 show up to a 1000-fold increased gene-targeting efficiency that is successfully harnessed to generate knockouts for a large number of genes. We inquired whether the increased gene-targeting efficiency of DT40 and NALM6 cells is linked to increased rates of HR-mediated DSB repair after exposure to ionizing radiation (IR). We analyzed IR-induced γ-H2AX foci as a marker for the total number of DSBs induced in a cell and RAD51 foci as a marker for the fraction of those DSBs undergoing repair by HR. We also evaluated RPA accretion on chromatin as evidence for ongoing DNA end resection, an important initial step for all pathways of DSB repair except c-NHEJ. We finally employed the DR-GFP reporter assay to evaluate DSB repair by HR in DT40 cells. Collectively, the results obtained, unexpectedly show that DT40 and NALM6 cells utilized HR for DSB repair at levels very similar to those of other somatic cells. These observations uncouple gene-targeting efficiency from HR contribution to DSB repair and suggest the function of additional mechanisms increasing gene-targeting efficiency. Indeed, our results show that analysis of the contribution of HR to DSB repair may not be used as a proxy for gene-targeting efficiency.

New Facets of DNA Double Strand Break Repair: Radiation Dose as Key Determinant of HR versus c-NHEJ Engagement

“…Results generated during the above study also support the notion that the suppression of HR activity at high IR doses is a regulated process, delimited but not defined by the 53BP1 and RAD52 activities [157]. The suppression of HR at high IR doses is not an isolated phenomenon, as a separate study utilizing a panel of somatic and lymphoblastic cell lines also demonstrated a pronounced suppression of HR at doses above 2 Gy [167]. Our results also indicated that the process of DNA end resection is uncoupled from HR in terms of IR dose-dependent regulation, as RPA foci formation saturates at much higher By calculating the ratio between RAD51 and γ-H2AX foci, we could evaluate the fraction of DSB engaging HR at any given IR dose (Figure 3C).…”

“…Similar PFGE experiments conducted with wild-type G 2 -phase-enriched cell lines, where HR activity is at a maximum, demonstrate repair capacity similar to that of exponentially growing cells [53]. The limited impact of HR on DSB repair in these experiments is further validated in experiments testing G 2 -phase cell populations exposed to a highly specific small molecule inhibitor, VE-821, which impedes HR through the abrogation of ATR activity [167]. Furthermore, we have reported that actively growing mouse embryonic fibroblasts (MEF) that are defective in HR repair, process DSBs with an efficiency similar to that of wild-type cells [169].…”

“…As outlined in the preceding sections, whether a specific DSB undergoes processing via c-NHEJ, HR, or alternative repair mechanisms is governed by various factors. Mounting evidence highlights the importance of radiation dose in the regulation of DSB repair pathway choice [157,167]. Interestingly, a study employing pulsed-field gel electrophoresis (PFGE) in mutant chicken cell lines lacking critical HR factors, Rad51, Rad51B, Rad52, and RAD54, allowed key insights into DSB repair regulation: there are no discernible differences in DSB repair kinetics among the investigated cell lines despite the grave HR defects.…”

“…To evaluate the contribution of HR to the repair of DSBs at more physiological IR doses, relevant to radiation protection and radiation therapy, we developed a cell-cycle specific immunofluorescence analysis of γ-H2AX foci, an established surrogate marker for DSB formation (Figure 3A) [170,171], in combination with the detection of RAD51 foci, as a marker for ongoing HR (Figure 3B) [172]. Figure 3A,B illustrate an idealized data set for γ-H2AX and RAD51 foci formation after IR exposure, based on our previously generated immunofluorescence analysis in G 2 -phase cells [157], where all DSB repair pathways are fully active [157,167,173]. Our results have shown that the maximum number of γ-H2AX foci is reached early after radiation exposure, irrespectively of the applied dose.…”

Drug Discovery Targeting Post-Translational Modifications in Response to DNA Damages Induced by Space Radiation

“…DSBs can be repaired by several organized mechanisms to maintain the stability and integrity of the genome [35], which is vital for cell survival. Classical nonhomologous end joining (NHEJ), homologous recombination (HR), alternative end joining (alt-EJ), and single-strand annealing (SSA) represent distinct DSB repair mechanisms [36], of which NHEJ and HR are the most pivotal and common. Post-translational modifications (PTMs) are covalent chemical modifications of proteins that occur after translation, conferring proper activity and biological functions to these proteins.…”