Bartosz Szczesny, Ph.D.
Integrity of the mitochondrial genome: the role of the DNA base excision repair pathway. Mitochondria are indispensable for cellular survival as the site of ATP generation and also regulator of apoptosis. Because of lack of chromatinization and close proximity to the site of oxidative phosphorylation, the mitochondrial (mt) genome is much more prone to oxidative damage than the nuclear. Oxidative damage due to reactive oxygen species (ROS) include oxidative base modifications, abasic (AP) sites and their oxidation products: single-strand breaks which are all repaired via the DNA base excision repair (BER) pathway in both nucleus and mitochondria; however, mt BER is much less understood. We recently identified the presence of long patch (LP)-BER in mitochondria and EXOG, a paralog of EndoG, a critical mt 5’ exo/edonuclease. Our focus currently is on biochemical characterization of EXOG, organization of mt BER machinery and identification of other proteins involved in mt genome repair.
Mitochondrial genome damage as a signal for intrinsic apoptosis. Single-strand breaks (SSBs), with non-ligatable 3’ and/or 5’ ends, cause genomic instability, stall RNA polymerases during transcription and generate highly toxic double-strand breaks during replication. Efficient SSB repair (SSBR), the sub-pathway of BER, is critical to maintaining cellular homeostasis, a process much less understand and studied in mitochondria than in its nuclear counterpart. The cellular effect of SSBs in the mt genome has yet to be determined. We are investigating the effect of persistent SSBs in mt DNA on mitochondria function and as an initial trigger in activating the intrinsic apoptotic pathway.
The role of single strand breaks in the mitochondrial genome in aging. Chronic oxidative stress causing damage to mitochondrial (mt) and nuclear genomes is generally accepted in the etiology of mammalian aging. The multi-step BER pathway maintains the integrity of both genomes. The essentiality of both abasic endonuclease 1 (APE1) and -- proposed recently by us -- 5’ exonuclease EXOG is likely due to their role in preventing oxidative stress and apoptosis. This suggests cellular toxicity caused by persistent unrepaired SSBs in the mt genome, rather than various types of oxidative lesions that accumulate with age. This idea is supported by the lack of severe phenotype of knockouts for several DNA glycosylases, the initial BER enzymes. We have proposed that persistent SSBs in the mt genome provides the initial trigger for multiple phenotypes of aging via stress-mediated signaling which we are currently investigating. As a model, sarcopenia (loss of muscle mass) is a hallmark of aging and we have shown that mouse skeletal muscles have limited BER capacity. We are currently examining the BER capacity of progenitor and terminally differentiated muscle cells and the role of mt genome integrity as a function of age and during the process of muscle differentiation.
