A Multi-Endpoint Approach to Base Excision Repair Incision Activity Augmented by PARylation and DNA Damage Levels in Mice: Impact of Sex and Age

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.

Ageing-associated effects of a long-term dietary modulation of four trace elements in mice

“…Especially with regard to the widespread theory of impaired DNA repair and accumulation of DNA damage as key factors causing and promoting ageing, this was unexpected [ 105 , 106 ]. Nevertheless, several human and rodent studies revealed comparable results with regard to DNA damage showing a missing correlation between increased DNA damage levels and age [ 51 , 107 , 108 ]. In line with the unaffected DNA damage levels, hepatic PARylation, key to DDR and BER [ 109 ], several mRNA expression levels of central BER proteins, as well as incision activities towards 8-oxodG, 5-OHdU, and an AP site analogue were not affected by diet and age ( Fig.…”

“…To detect single- and double-strand breaks as well as alkali-labile sites, the alkaline comet assay was performed [ 50 ]. Cell isolation from 40 mg of frozen liver tissue, experimental procedure, and final analysis of 200 randomly selected cells (100 per technical replicate) by semi-automated image analysis software (Comet IV, Perceptive Instruments, Stone, UK) were carried out as previously published [ 51 ], following the OECD guideline 489 and the recommendations for minimum information for reporting on the comet assay (MICRA) [ [51] , [52] , [53] ].…”

“…BER incision activity was assessed as previously described [ 51 ]. In brief, 50 mg of snap-frozen liver tissue were employed for tissue extract preparation.…”

“…Analysis of PARylation levels was performed via HPLC-MS/MS (HPLC: 1260 Infinity, Agilent; MS/MS: 6495A, Agilent) after extraction of poly(ADP-ribose) (PAR) from murine liver tissue according to previously published protocols [ 51 , 54 ]. Briefly, 10–20 mg of liver tissue were applied for PAR analysis.…”

“…Protein extracts were prepared as previously described [ 51 ] for BER incision activity assay, diluted 1:3 with 4x Laemmli sample buffer (0.25 M Tris pH 6.8, 8 % SDS, 40 % Glycerol, 0.03 % bromophenol blue) followed by denaturation for 5 min at 95°C, and stored at -80°C until further use [ 51 ]. Proteins were loaded and separated on 15 % polyacrylamide gels and transferred to a 0.45 μm nitrocellulose membrane (Amersham Protran, Merck (Sigma-Aldrich)) via tank blotting (Bio-Rad Laboratories).…”