Poly(ADP-ribosyl)ation and DNA repair synthesis in the extracts of naked mole rat, mouse, and human cells

Anastasiya A. Kosova,Ekaterina A. Belousova,Ekaterina S. Ilina,Mikhail M. Kutuzov,Svetlana A. Romanenko,Alexei N. Evdokimov,Vladimir A. Trifonov,Olga I. Lavrik,Svetlana N. Khodyreva

doi:10.18632/aging.101959

Abstract

DNA repair capacity in cells of naked mole rat (Hgl), a species known for its longevity and resistance to cancer, is still poorly characterized. Here, using the whole-cell extracts (WCEs) of Hgl, mouse and human cells, we studied the interrelation between DNA synthesis on the substrates of base excision repair and the activity of poly(ADP-ribose) polymerases (PARPs) responsible for the transfer of the ADP-ribose moieties onto different targets. The level of PAR synthesis was more than ten-fold higher in human WCE as compared to rodent WCEs, while the efficiency of DNA synthesis was comparable. Under conditions of PAR synthesis, the efficiency of DNA synthesis was only slightly enhanced in all extracts and in mouse WCEs unusual products of the primer elongation were detected. The results obtained with WCEs, recombinant proteins and recently found ability of PARPs to attach the ADP-ribose moieties to DNA allowed us to attribute these products to primer mono(ADP-ribosyl)ation (MARylation) at the 5ʹ-terminal phosphate by PARP3 during the DNA synthesis. PARP1/PARP2 can then transfer the ADP-ribose moieties onto initial ADP-ribose. Our results suggest that MARylation/PARylation of DNA in the extracts depends on the ratios between PARPs and can be controlled by DNA-binding proteins.

Highlights

DNA repair systems are considered as a key factor in mammalian cells, which counteracts genomic instability and is associated with aging and oncogenesis
The changes occurred almost to the same extent in all extracts irrespectively of both the total efficiency of PAR synthesis (Figure 1) and the amount of PARP1, which was evaluated by crosslinking of the extract proteins with the photoreactive base excision repair (BER) substrates (Figure 6, lanes 4–12)
Earlier, using a system reconstituted from purified proteins and bovine testis nuclear extract supplemented with recombinant PARP1 we have revealed that PARP1 may act as a regulator of polymerase β (Polβ) activity in the LP-BER pathway and that PARylation of PARP1 plays a crucial role in ensuring of the Polβ-mediated DNA synthesis [17, 40]

Summary

Introduction

DNA repair systems are considered as a key factor in mammalian cells, which counteracts genomic instability and is associated with aging and oncogenesis. Until recently DNA repair efficiency in Hgl cells has not been directly tested. Using extracts from Hgl and mouse (Mus musculus, Mmu) fibroblasts we compared the activities of some enzymes involved in base excision repair (BER) [1]. Hgl cell extracts were slightly more efficient compared to Mmu in the removal of the uracil residues and cleavage of the abasic (AP) sites but not in the DNA synthesis on the BER substrates. The level of the poly(ADP-ribose) (PAR) synthesis catalyzed by poly(ADP-ribose) polymerases (PARPs) was higher in Hgl cell extracts. Hgl cell extracts contain higher amounts of PARP1 as revealed by crosslinking of the extract proteins to chemically reactive www.aging-us.com

Methods

Results

Conclusion