Complementation of aprataxin deficiency by base excision repair enzymes in mitochondrial extracts

Abstract

Mitochondrial aprataxin (APTX) protects the mitochondrial genome from the consequence of ligase failure by removing the abortive ligation product, i.e. the 5′-adenylate (5′-AMP) group, during DNA replication and repair. In the absence of APTX activity, blocked base excision repair (BER) intermediates containing the 5′-AMP or 5′-adenylated-deoxyribose phosphate (5′-AMP-dRP) lesions may accumulate. In the current study, we examined DNA polymerase (pol) γ and pol β as possible complementing enzymes in the case of APTX deficiency. The activities of pol β lyase and FEN1 nucleotide excision were able to remove the 5′-AMP-dRP group in mitochondrial extracts from APTX−/− cells. However, the lyase activity of purified pol γ was weak against the 5′-AMP-dRP block in a model BER substrate, and this activity was not able to complement APTX deficiency in mitochondrial extracts from APTX−/−Pol β−/− cells. FEN1 also failed to provide excision of the 5′-adenylated BER intermediate in mitochondrial extracts. These results illustrate the potential role of pol β in complementing APTX deficiency in mitochondria.

APE1: A skilled nucleic acid surgeon

“…In addition, the APE1 exonuclease activity occurs at a similar rate to the activities of other enzymes in the BER pathway ( Table 1 ). Variety in the type of end blocking group and the assortment of possible DNA termini (i.e., nicks, gaps, overhangs, and double strand breaks) in need of processing during multiple different repair pathways, further combined with the existence of multiple end processing enzymes with varying substrate specificities has resulted in a murky picture of the exact biological role of each end processing enzyme, and underscores the importance of further characterizing these enzymes and their activities [ 37 , [40] , [41] , [42] , [43] , [44] , [45] , [46] , [47] , [48] , [49] , [50] , [51] , [52] , [53] ]. This substrate overlap implies a level of evolved redundancy among enzymes that may play a particular biological role during different cellular stages and in response to varying types of DNA damage.…”

Autosomal Recessive Cerebellar Ataxias

“…It seems to be more critical in mitochondrial DNA repair than in the nuclear DNA repair, and its deficiency associates with mitochondrial abnormalities including mitochondrial morphology and network. No specific treatment approaches are available yet (Akbari et al 2015;Caglayan et al 2017). As mitochondria have become targets for pharmacological interventions for treatment of neurodegenerative diseases with promising results, such strategies should be then tested on AOA1 patients.…”

DNA ligase I fidelity mediates the mutagenic ligation of pol β oxidized nucleotide insertion products and base excision repair intermediates with mismatches

“…Oligodeoxyribonucleotides with and without a 6-carboxyfluorescein (FAM) label and the 5'adenylate (AMP) were obtained from Integrated DNA Technologies. The DNA substrates used in this study were prepared as described previously (12)(13)(14)(15)(16)(34)(35)(36)38,39). The one nucleotide gapped DNA substrates were used for coupled assays (Supplementary Table 1).…”

“…Aprataxin (APTX) and flap endonuclease 1 (FEN1) activity analyses were performed as described previously (34)(35)(36). For this purpose, we used the nicked DNA substrates containing 5'-AMP and 3'-preinserted damaged (8-oxodG) or all possible 12 mismatched bases (Supplementary Table 3).…”

“…Aprataxin (APTX), a member of the histidine triad (HIT) superfamily, hydrolyzes an adenylate (AMP) moiety from 5'-end of ligation failure products and allows further attempts at completing repair (33). In our previous studies, we reported a role of Flap Endonuclease 1 (FEN1) for the processing of blocked repair intermediates with 5'-AMP (34)(35)(36). This compensatory mechanism is important to complement a deficiency in the APTX activity, which is associated with the mutations in the aptx gene and linked to the autosomal recessive neurodegenerative disorder Ataxia with oculomotor apraxia type 1 (AOA1) (37).…”