Understanding the Mechanism of DNA Double-Strand Break (DSB) Repair in Mitochondria and Various Mammalian Organs.

Abstract

Various exogenous and endogenous agents can damage DNA. Exogenous agents include chemicals and radiation, such as ionizing radiations and UV rays, while endogenous agents include DNA replication errors, reactive oxygen species (ROS), viruses, and transposons. These agents can cause various types of DNA damage, such as base modifications, cross-linking, and strand or double-strand breaks. Among these, DSBs are the most lethal form of DNA damage, leading to consequences such as loss of heterozygosity, mutations, cancer, and even cell death.

DSBs can be repaired through several mechanisms: Homologous Recombination (HR), Non-Homologous End Joining (NHEJ), Microhomology-Mediated End Joining (MMEJ), and Single-Strand Annealing (SSA). HR is has high fidelity, uses a homologous template for repair. HR is mainly active during the S and G2 phases. NHEJ, a faster but error-prone mechanism, directly ligates broken ends. It can occurs throughout the cell cycle, with primary activity in the G1 phase. SSA and MMEJ are error-prone DNA repair mechanisms for DSBs; SSA occurs when DSBs are flanked by repetitive sequences, leading to the deletion of the intervening region after annealing of single-stranded DNA. It requires longer microhomology sequences and Rad52 for their annealing, whereas MMEJ require short microhomologous sequences and involves Pol θ and PARP1, which are crucial for MMEJ. Despite reported differences, the distinction between MMEJ and SSA remains unclear, and it is still uncertain whether they represent separate pathways or share a common mechanism. This ambiguity continues to be an active area of research.

Unlike HR, MMEJ is error-prone and linked to genomic instability, contributing to cancers and neurological diseases. MMEJ has also been observed in mitochondrial disorders. Though traditionally thought to occur without cNHEJ factors, recent studies suggest that MMEJ can occur in normal cells, even when cNHEJ and HR are functional.

In the present study, we examined MMEJ in normal tissues during non-pathological processes, focusing on its dependence on microhomology length and age. We have used an MMEJ substrate simulating double-strand breaks (DSBs) flanked by direct repeats of 10 to 22 nt. The results revealed the highest MMEJ activity in the thymus, spleen, and testes and the lowest in the brain, lungs, and kidneys. Time kinetics showed rapid MMEJ in the thymus, spleen, and testes. At the same time, minimal activity was observed in the lungs and kidneys after 4 h. Increasing microhomology length (from 10 to 16-22 nt) shifted MMEJ activity, with the lungs exhibiting the highest activity at longer microhomologies. A sharp decline in MMEJ in the spleen was noted as microhomology length increased. Notably, the thymus maintained efficient MMEJ across all microhomology lengths.

Differential MMEJ activity correlated with the expression of key MMEJ proteins (PARP1, Pol θ, MRE11, Fen1, Lig3-Xrcc1). The thymus, spleen, and testes exhibited high MMEJ with shorter microhomology substrates and elevated protein expression, while the lungs and kidneys showed low MMEJ with shorter but higher activity with longer microhomology substrates. The brain's lack of MMEJ with shorter microhomology was attributed to the absence of PARP1, highlighting its critical role in MMEJ.

Our study also explored age-related changes in MMEJ across organs. In early-aged rats, minimal MMEJ was observed in the lungs and kidneys with short microhomology, but significant increases were noted with ageing. Age-related increases in MMEJ were observed in the testes, lungs, kidneys, and liver, while decreases were seen in the spleen and thymus. These changes were linked to BRCA1, Ligase III, PARP1, and Cdk2 expression alterations, suggesting age-related genomic instability.

Next, we investigated the role of MMEJ in mitochondrial genome instability. Large deletions in the mitochondrial genome were observed; direct repeats flank most deletions. Our interest lies in understanding the physiological conditions that drive the observed deletions. As mitochondria are the primary source of ROS, we tested the contribution of aberrant ROS on mitochondrial DNA repair. To mimic elevated ROS, we used menadione, a chemical that induces heightened ROS production by futile redox cycling for 1h, followed by mitochondria extract (ME) preparation.

The results showed an increase in MMEJ following menadione treatment and forming a 50-bp joined product, which was absent in the untreated control. This suggests that menadione treatment upregulates additional DNA repair pathways to mitigate damage caused by reactive ROS. In addition to MMEJ, a significant increase in homologous recombination (HR) was also observed. The mechanism underlying this increase in DSB repair included a significant rise in the localization of DNA ligase III and MRE11 in mitochondria, with a non-significant increase in PARP1 and no change in RAD51 levels. Additionally, daily exposure to ionizing radiation (IR) through medical, natural, or occupational sources induces DSBs in both the nucleus and mitochondria, with more severe and prolonged damage in mitochondria. Since mitochondria lack sufficient DNA repair proteins, they rely on nuclear repair mechanisms. Upon exposure to 2 Gy and 5 Gy IR, MMEJ in mitochondria decreased, likely due to increased MMEJ activity in the nucleus. Mitochondrial DNA damage was primarily repaired via HR, with IR exposure enhancing HR activity. Following IR, mitochondrial repair involved a significant increase in DNA ligase III but a non-significant decrease in MRE11. However, the increase in DNA ligase III did not correspond with the decrease in MMEJ, suggesting that mitochondria may compensate for DNA damage by elevating DNA replication facilitated by an increase in enhanced ligase III. These findings emphasize the dynamic interplay between various DNA repair mechanisms in response to IR-induced damage in mitochondria.

This study examined the role of Microhomology-Mediated End Joining (MMEJ) in normal tissues and mitochondrial genome instability, focusing on its dependence on microhomology length and age. MMEJ activity varied across organs, with the thymus, spleen, and testes showing the highest activity and the brain, lungs, and kidneys the lowest. Age-related changes in MMEJ were observed, with increases in the testes, lungs, kidneys, and liver and decreases in the spleen and thymus. The study also investigated the impact of ROS on mitochondrial DNA repair, revealing that menadione-induced ROS elevate both MMEJ and homologous recombination (HR). Following ionizing radiation (IR) exposure, mitochondrial MMEJ decreased while HR activity increased, underscoring a compensatory shift in DNA repair mechanisms. These results underscore the dynamic interaction between DNA repair pathways in response to oxidative and radiation-induced damage.