Identification and biochemical analysis of a mitochondrial endonuclease of Podospora anserina related to curved-DNA binding proteins

Laquel-Robert, Patricia; Sellem, Carole H.; Sainsard-Chanet, Annie; Castroviejo, Michel

doi:10.1016/j.bbagen.2006.10.003

Cited by 1 publication

(5 citation statements)

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“…However, the radiola- beled 31-nt band, observed after [␣-32 P]dAMP incorporation suggests resynthesis of the template 5Ј to the THF site, which is removed by the 3Ј-exonuclease activity of pol ␥ ( Table 1). Radiolabeling of the 52-nt band by [␣- 32 P]dCMP and [␣-32 P]dTMP indicated repair due to replacement of the nucleotides on both sides of the THF (Fig. 2F) 32 P]dTMP were likely to represent incomplete synthesis after exonucleolytic degradation by pol ␥, and similar fragments were observed after [␣-…”

Section: Sn-ber and Lp-ber Activities In Mitochondrial Extracts-mentioning

confidence: 88%

“…We have detected a 5Ј-exo/endonuclease activity distinct from FEN-1, and further characterization of this activity will be required. It is worth noting that the mitochondria of Podospora anserine express a 5Ј-exonuclease with sequence homology to yeast RAD2 (an ortholog of FEN-1), which is active on various substrates including DNA with a flap (31,32). Finally, while mitochondrial FEN-1 could be dispensable for BER, it may be still required for 5Ј terminus processing of Okazaki fragments during mitochondrial replication (33,34).…”

Section: Discussionmentioning

confidence: 99%

“…3Ј-End labeling was performed for an oligonucleotide with the same sequence as oligo5 but one nucleotide shorter at the 3Ј terminus. After annealing with the complementary oligo, the 1-nt 5Ј-overhang was used as the template for filling with [␣- 32 P]dTMP at the 3Ј-end using 3Ј-exo-deficient DNA polymerase (Klenow Fragment, New England BioLabs).…”

Section: Methodsmentioning

confidence: 99%

“…To characterize LP-BER activity of mitochondrial extracts, the repair of THF-containing oligo5 labeled at the 5Ј or 3Ј terminus with 32 P was carried out (Table 1). First, we analyzed the size of 5Ј-end-labeled oligo4, which does not contain any damage, after its incubation with mitochondrial extract of young liver, and we could not detect any qualitative changes ( Fig.…”

Section: Sn-ber and Lp-ber Activities In Mitochondrial Extracts-mentioning

confidence: 99%

“…Incorporation of multiple nucleotides in the THF oligo substrate could thus be due to the filling of gap generated by the 3Ј-exonuclease activity of pol ␥ or a 5Ј-exo/endonuclease, such as FEN-1, or both. To establish the directionality of repair synthesis, we designed the sequence of oligo6 ( 32 P]dGMP at the THF site after strand cleavage. Persistence of the cleaved and radiolabeled fragment strongly suggests that its ligation or 3Ј-exonucleolytic processing of the downstream fragment was rate-limiting.…”

Section: Sn-ber and Lp-ber Activities In Mitochondrial Extracts-mentioning

confidence: 99%

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Long Patch Base Excision Repair in Mammalian Mitochondrial Genomes

Szczesny

Tann

Longley

et al. 2008

Journal of Biological Chemistry

146

138

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The mitochondrial genome is highly susceptible to damage by reactive oxygen species (ROS) generated endogenously as a byproduct of respiration. ROS-induced DNA lesions, including oxidized bases, abasic (AP) sites, and oxidized AP sites, cause DNA strand breaks and are repaired via the base excision repair (BER) pathway in both the nucleus and mitochondria. Repair of damaged bases and AP sites involving 1-nucleotide incorporation, named single nucleotide (SN)-BER, was observed with mitochondrial and nuclear extracts. During SN-BER, the 5-phosphodeoxyribose (dRP) moiety, generated by AP-endonuclease (APE1), is removed by the lyase activity of DNA polymerase ␥ (pol ␥) and polymerase ␤ in the mitochondria and nucleus, respectively. However, the repair of oxidized deoxyribose fragments at the 5 terminus after strand break would require 5-exo/endonuclease activity that is provided by the flap endonuclease (FEN-1) in the nucleus, resulting in multinucleotide repair patch (long patch (LP)-BER). Here we show the presence of a 5-exo/endonuclease in the mitochondrial extracts of mouse and human cells that is involved in the repair of a lyase-resistant AP site analog via multinucleotide incorporation, upstream and downstream to the lesion site. We conclude that LP-BER also occurs in the mitochondria requiring the 5-exo/endonuclease and pol ␥ with 3-exonuclease activity. Although a FEN-1 antibody cross-reacting species was detected in the mitochondria, it was absent in the LP-BER-proficient APE1 immunocomplex isolated from the mitochondrial extract that contains APE1, pol ␥, and DNA ligase 3. The LP-BER activity was marginally affected in FEN-1-depleted mitochondrial extracts, further supporting the involvement of an unidentified 5-exo/endonuclease in mitochondrial LP-BER.The mammalian mitochondrion contains 5-15 copies of the circular 16-kb mitochondrial (mt) 2 genome, and each mammalian cell thus may contain a thousand or more copies of the mt genome (1). MtDNA, encoding 13 subunits of the electron transport chain and containing genes for ribosomal RNAs and tRNAs (2), is extremely susceptible to oxidative damage, presumably because of the lack of protective histones and proximity to reactive oxygen species (ROS), which are endogenously generated by the electron transport complexes (3, 4). Such damage includes several dozen oxidized bases, abasic (AP) sites, and oxidation products of AP sites leading to DNA strand breaks (5). Endogenous mutations in mtDNA, likely to arise from these lesions, were shown to be considerably higher than in the nuclear genome (6). Approximately 10,000 AP sites were estimated to be generated per nuclear genome per day (7). Analysis of the release of 5-methylene-2-furanone, the product of ␤-, ␥-elimination of 2-deoxyribonolactone, an oxidized AP site, causing DNA strand breakage, suggests that this ribonolactone could account for 70% of the total sugar damage in DNA (8, 9). The oxidized AP sites, whose level is likely to be high especially in the mtDNA, should block replication and transcription a...

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Section: Sn-ber and Lp-ber Activities In Mitochondrial Extracts-mentioning

confidence: 88%