Abstract
Investigation of processes that contribute to the maintenance of genomic stability is one crucial factor in the attempt to understand mechanisms that facilitate ageing. The DNA damage response (DDR) and DNA repair mechanisms are crucial to safeguard the integrity of DNA and to prevent accumulation of persistent DNA damage. Among them, base excision repair (BER) plays a decisive role. BER is the major repair pathway for small oxidative base modifications and apurinic/apyrimidinic (AP) sites. We established a highly sensitive non-radioactive assay to measure BER incision activity in murine liver samples. Incision activity can be assessed towards the three DNA lesions 8-oxo-2’-deoxyguanosine (8-oxodG), 5-hydroxy-2’-deoxyuracil (5-OHdU), and an AP site analogue. We applied the established assay to murine livers of adult and old mice of both sexes. Furthermore, poly(ADP-ribosyl)ation (PARylation) was assessed, which is an important determinant in DDR and BER. Additionally, DNA damage levels were measured to examine the overall damage levels. No impact of ageing on the investigated endpoints in liver tissue were found. However, animal sex seems to be a significant impact factor, as evident by sex-dependent alterations in all endpoints investigated. Moreover, our results revealed interrelationships between the investigated endpoints indicative for the synergetic mode of action of the cellular DNA integrity maintaining machinery.
Highlights
The limited chemical stability of DNA under physiological conditions facilitates the constant formation of a broad spectrum of DNA lesions [1]
By the combined assessment of base excision repair (BER) incision activity, the PARylation status, and DNA damage levels in the liver of adult and old mice of both sexes, this paper aims to contribute to a deeper understanding of the impact of age and sex on the genomic stability maintaining machinery
We added the non-ionic surfactant Triton X-100, known to improve cellular membrane permeability [41], in a final concentration of 1% to the extraction buffer. This enhances the release of the soluble protein fraction from the tissue-bound liver cells that have a more complex matrix than cultured cells
Summary
The limited chemical stability of DNA under physiological conditions facilitates the constant formation of a broad spectrum of DNA lesions [1]. DNA lesions can have deleterious consequences, that eventually can cause cancer and accelerate ageing [2]. Nuclear DNA damage accumulation and genomic instability seem to be important drivers in ageing, leading to cellular functional decline or loss of tissue homeostasis [3,4]. Cellular mechanisms including the DNA damage response (DDR) and DNA repair are essential [2,5]. According to the general opinion stated in the literature, DNA damage increases with age, whereas the DNA repair capacity declines with age [5]. The major cellular repair pathway to remove the vast and frequently occurring spectrum of small oxidative base modifications, apurinic/apyrimidinic (AP) sites, and single-strand breaks is base excision repair (BER) (reviewed in [6,7,8,9,10])
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