Efficiency of nonhomologous DNA end joining varies among somatic tissues, despite similarity in mechanism

Abstract

Failure to repair DNA double-strand breaks (DSBs) can lead to cell death or cancer. Although nonhomologous end joining (NHEJ) has been studied extensively in mammals, little is known about it in primary tissues. Using oligomeric DNA mimicking endogenous DSBs, NHEJ in cell-free extracts of rat tissues were studied. Results show that efficiency of NHEJ is highest in lungs compared to other somatic tissues. DSBs with compatible and blunt ends joined without modifications, while noncompatible ends joined with minimal alterations in lungs and testes. Thymus exhibited elevated joining, followed by brain and spleen, which could be correlated with NHEJ gene expression. However, NHEJ efficiency was poor in terminally differentiated organs like heart, kidney and liver. Strikingly, NHEJ junctions from these tissues also showed extensive deletions and insertions. Hence, for the first time, we show that despite mode of joining being generally comparable, efficiency of NHEJ varies among primary tissues of mammals.

Time-Dependent Predominance of Nonhomologous DNA End-Joining Pathways during Embryonic Development in Mice

“…15,39,50,53,54 In contrast to some of the earlier reports, we also observed intramolecular circularization. 42,[53][54][55] Comparison of NHEJ activities showed that the joining of DSBs was highest in 14.5-day cephalon, irrespective of the nature of DSBs. Consistent with this, immunofluorescence studies using p53BP1 also showed the highest levels of innate DSBs within the cells at this stage, justifying the need for an efficient NHEJ system.…”

“…[42][43][44][45][46] Besides the efficiency of repair, the mechanism of regulation of NHEJ, the regulation of their proteins, and various protein-protein interactions were also studied using such a system. 15,39,[43][44][45][47][48][49][50][51][52][53][54] However, it is not clear whether the end-joining events observed in our studies correspond to in vivo NHEJ events, although the various lines of experimental evidence suggest an in vivo mechanistic correlation. Moreover, we could also correlate the expression of proteins detected by immunoblotting at different developmental stages to the immunofluorescence observed at the intracellular level (see the text below).…”

“…3). 39,48,54 Previous studies also suggest that complementary cohesive ends can be rejoined in an error-free manner by precise ligation and may require only some components such as KU proteins and the XRCC4-ligase IV complex of the NHEJ pathway. 56 In contrast, rejoining of noncompatible DNA ends requires end processing and thus additional factors such as Artemis, DNA-PKcs, and the polymerase activities of Pol μ and Pol λ.…”

Mechanism of Fragility at BCL2 Gene Minor Breakpoint Cluster Region during t(14;18) Chromosomal Translocation

“…The oligomers were gel-purified as described (37). The 5Ј end labeling of the oligomeric DNA was done using T4 polynucleotide kinase with [␥-32 P]ATP (38). For details of preparation of oligonucleotide DNA substrates, refer to the supplemental Materials and Methods.…”

Structure‐specific nuclease activity of RAGs is modulated by sequence, length and phase position of flanking double‐stranded DNA

“…5 0 end labeling of oligomeric DNA T4 DNA polynucleotide kinase was used for 5 0 end labeling of the oligomeric DNA, and labeling was done as described previously [31,33]. The labeled substrates were purified using a Sephadex G-25 column and stored at À20°C until use.…”