Membrane association of mitochondrial DNA facilitates base excision repair in mammalian mitochondria

Mitochondrial DNA encodes a set of 13 polypeptides and is subjected to constant oxidative stress due to ROS production within the organelle. It has been shown that DNA repair in the mitochondrion proceeds through both short- and long-patch base excision repair (BER). In the present article, we have used the natural competence of mammalian mitochondria to import DNA and study the sub-mitochondrial localization of the repair system in organello. Results demonstrate that sequences corresponding to the mtDNA non-coding region interact with the inner membrane in a rapid and saturable fashion. We show that uracil containing import substrates are taken into the mitochondrion and are used as templates for damage driven DNA synthesis. After further sub-fractionation, we show that the length of the repair synthesis patch differs in the soluble and the particulate fraction. Bona fide long patch BER synthesis occurs on the DNA associated with the particulate fraction, whereas a nick driven DNA synthesis occurs when the uracil containing DNA accesses the soluble fraction. Our results suggest that coordinate interactions of the different partners needed for BER is only found at sites where the DNA is associated with the membrane.     

Role of DNA polymerase beta in the excision step of long patch mammalian base excision repair.

The two base excision repair (BER) subpathways in mammalian cells are characterized by the number of nucleotides synthesized into the excision patch. They are the "single-nucleotide" BER pathway and the "long patch" (several nucleotides incorporated) BER pathway. Both of these subpathways involve excision of a damaged base and/or nearby nucleotides and DNA synthesis to fill the excision gap. Whereas DNA polymerase beta (pol beta) is known to participate in the single-nucleotide BER pathway, the identity of polymerases involved in long patch BER has remained unclear. By analyzing products of long patch excision generated during BER of a uracil-containing DNA substrate in mammalian cell extracts we find that long patch excision depends on pol beta. We show that the excision of the characteristic 5'-deoxyribose phosphate containing oligonucleotide (dRP-oligo) is deficient in extracts from pol beta null cells and is rescued by addition of purified pol beta. Also, pol beta-neutralizing antibody inhibits release of the dRP-oligo in wild-type cell extracts, and the addition of pol beta after inhibition with antibody completely restores the excision reaction. The results indicate that pol beta plays an essential role in long patch BER by conducting strand displacement synthesis and controlling the size of the excised flap.     

Single-nucleotide patch base excision repair of uracil in DNA by mitochondrial protein extracts.

Mammalian mitochondria contain several 16.5 kb circular DNAs (mtDNA) encoding electron transport chain proteins. Reactive oxygen species formed as byproducts from oxidative phosphorylation in these organelles can cause oxidative deamination of cytosine and lead to uracil in mtDNA. Upon mtDNA replication, these lesions, if unrepaired, can lead to mutations. Until recently, it was thought that there was no DNA repair in mitochondria, but lately there is evidence that some lesions are efficiently repaired in these organelles. In the study of nuclear DNA repair, the in vitro repair measurements in cell extracts have provided major insights into the mechanisms. The use of whole-cell extract based DNA repair methods has revealed that mammalian nuclear base excision repair (BER) diverges into two pathways: the single-nucleotide replacement and long patch repair mechanisms. Similar in vitro methods have not been available for the study of mitochondrial BER. We have established an in vitro DNA repair system supported by rat liver mitochondrial protein extract and DNA substrates containing a single uracil opposite to a guanine. Using this approach, we examined the repair pathways and the identity of the DNA polymerase involved in mitochondrial BER (mtBER). Employing restriction analysis of in vitro repaired DNA to map the repair patch size, we demonstrate that only one nucleotide is incorporated during the repair process. Thus, in contrast to BER in the nucleus, mtBER of uracil in DNA is solely accomplished by single-nucleotide replacement.     

FEN1 stimulation of DNA polymerase beta mediates an excision step in mammalian long patch base excision repair

In mammalian cells, single-base lesions, such as uracil and abasic sites, appear to be repaired by at least two base excision repair (BER) subpathways: "single-nucleotide BER" requiring DNA synthesis of just one nucleotide and "long patch BER" requiring multi-nucleotide DNA synthesis. In single-nucleotide BER, DNA polymerase beta (beta-pol) accounts for both gap filling DNA synthesis and removal of the 5'-deoxyribose phosphate (dRP) of the abasic site, whereas the involvement of various DNA polymerases in long patch BER is less well understood. Recently, we found that beta-pol plays a role in mammalian cell extract-mediated long patch BER, in that formation of a key excision product, 5'-dRP-trinucleotide (5'-dRP-N(3)), is dependent upon beta-pol (Dianov, G. L., Prasad, R., Wilson, S. H., and Bohr, V.A. (1999) J. Biol. Chem. 274, 13741-13743). The structure-specific endonuclease flap endonuclease 1 (FEN1) has also been suggested to be involved in long patch BER excision. Here, we demonstrate by immunodepletion experiments that 5'-dRP-N(3) excision in long patch BER of uracil-DNA in a human lymphoid cell extract is, indeed, dependent upon FEN1. Next, we reconstituted the excision step of long patch BER using purified human proteins and an oligonucleotide substrate with 5'-dRP at the margin of a one-nucleotide gap. Formation of the excision product 5'-dRP-N(3) was dependent upon both strand displacement DNA synthesis by beta-pol and FEN1 excision. FEN1 stimulated strand displacement DNA synthesis of beta-pol. FEN1 acting either alone, or without DNA synthesis by beta-pol, produced a two-nucleotide excision product, 5'-dRP-N(1), but not 5'-dRP-N(3). These results demonstrate that human FEN1 and beta-pol can cooperate in long patch BER excision and specify the predominant excision product seen with a cell extract.     

Long-patch DNA repair synthesis during base excision repair in mammalian cells

The base excision repair (BER) process removes base damage such as oxidation, alkylation or abasic sites. Two BER sub-pathways have been characterized using in vitro methods, and have been classified according to the length of the repair patch as either 'short-patch' BER (one nucleotide) or 'long-patch' BER (LP-BER; more than one nucleotide). To investigate the occurrence of LP-BER in vivo, we developed an assay using a plasmid containing a single modified base in the transcribed strand of the enhanced green fluorescent protein (EGFP) gene and a stop codon, based on a single-nucleotide mismatch, at varying distances on the 3' side of the lesion. The reversion of the stop codon occurs after DNA repair synthesis and restores EGFP expression after transfection of mismatch-repair-deficient cells. Repair patches longer than one nucleotide were observed for 55-80% or 80-100% of the plasmids with a mean length of 2-6 or 6-12 nucleotides for 8-oxo-7,8-dihydroguanine or a synthetic abasic site, respectively. These data show the existence of LP-BER in vivo, and emphasize the effect of the type of BER substrate lesion on both the yield and the extent of the LP-BER sub-pathway.     

HMGB1 is a cofactor in mammalian base excision repair

Deoxyribose phosphate (dRP) removal by DNA polymerase beta (Pol beta) is a pivotal step in base excision repair (BER). To identify BER cofactors, especially those with dRP lyase activity, we used a Pol beta null cell extract and BER intermediate as bait for sodium borohydride crosslinking. Mass spectrometry identified the high-mobility group box 1 protein (HMGB1) as specifically interacting with the BER intermediate. Purified HMGB1 was found to have weak dRP lyase activity and to stimulate AP endonuclease and FEN1 activities on BER substrates. Coimmunoprecipitation experiments revealed interactions of HMGB1 with known BER enzymes, and GFP-tagged HMGB1 was found to accumulate at sites of oxidative DNA damage in living cells. HMGB1(-/-) mouse cells were slightly more resistant to MMS than wild-type cells, probably due to the production of fewer strand-break BER intermediates. The results suggest HMGB1 is a BER cofactor capable of modulating BER capacity in cells.     

Protein-protein interactions and posttranslational modifications in mammalian base excision repair

Base excision repair (BER) averts the cytotoxic and mutagenic effects of most endogenously produced DNA damage, including lesions that arise spontaneously due to the intrinsic instability of DNA or modifications that are formed from reactions with intracellular chemicals, such as reactive oxygen species and alkylating agents. Defects in the BER process have been associated with cancer susceptibility and neurodegenerative disorders. In its most simplistic form, BER can be fully reconstituted with a minimum of four human proteins and is completed in just five sequential steps: (i) excision of an inappropriate base by a DNA glycosylase (e.g., uracil DNA glycosylase); (ii) incision of the DNA backbone immediately adjacent to the resulting abasic site by apurinic/apyrimidimic endonuclease 1; (iii) removal of the 5'-abasic terminal fragment, and (iv) repair synthesis to fill the gap by DNA polymerase beta; and (v) ligation to seal the remaining nick by DNA ligase 1 or a complex of DNA ligase 3 and X-ray repair cross-complementing 1. However, BER can involve the participation of other proteins as well, such as alternative DNA polymerases or one of several nonessential "auxiliary" factors. In addition, BER operates most efficiently when specific protein-protein coordination occurs. Furthermore, several BER protein activities have been shown to be regulated by posttranslational modification, and some of the physical protein interactions link BER to other DNA transaction pathways. In this review, we summarize the current state of the emerging complexities of mammalian BER, focusing on the growing number of reported protein-protein interactions and posttranslational modifications.     

ATP-dependent selection between single nucleotide and long patch base excision repair

DNA base excision repair (BER) constitutes a major mechanism to restore the integrity of the genome following modifications of nucleobases. Although it is well established that poly(ADP-ribosylation) facilitates BER, the mechanism of this stimulation has remained unknown. Previous observations suggested that poly(ADP-ribose), which is synthesised from NAD(+), could serve as a unique source of ATP required for the ligation step in BER. This pathway of ATP generation is thought to compensate ATP shortage and relies on the release of pyrophosphate during DNA repair synthesis. Here, we present evidence that, in situations of cellular energy depletion, the synthesis of poly(ADP-ribose) is indeed stimulated. Simultaneously, single nucleotide repair is reduced. Rather, the number of nucleotides incorporated by DNA polymerase beta (Pol beta) during DNA repair synthesis is increased. Using a reconstituted system including the recombinant BER proteins Pol beta, AP endonuclease 1 (APE 1), X-ray repair cross-complementing group-1 (XRCC1), DNA ligase III (Lig III), flap endonuclease 1 (FEN 1), and poly(ADP-ribose) polymerase-1 (PARP-1), it is demonstrated that in the absence of ATP, both long patch DNA synthesis by Pol beta and poly(ADP-ribosylation) catalysed by PARP-1 are stimulated. Consequently, the preferred use of either long patch or single nucleotide BER depends on the availability of ATP. It is proposed that long patch BER is required for ATP generation from poly(ADP-ribose) and, therefore, predominant under conditions of ATP shortage.     

Exchangeability of mammalian DNA ligases between base excision repair pathways

In mammalian cells, DNA ligase IIIalpha and DNA ligase I participate in the short- and long-patch base excision repair pathways, respectively. Using an in vitro repair assay employing DNA ligase-depleted cell extracts and DNA substrates containing a single lesion repaired either through short-patch (regular abasic site) or long-patch (reduced abasic site) base excision repair pathways, we addressed the question whether DNA ligases are specific to each pathway or if they are exchangeable. We find that immunodepletion of DNA ligase I did not affect the short-patch repair pathway but blocked long-patch repair, suggesting that DNA ligase IIIalpha is not able to substitute DNA ligase I during long-patch repair. In contrast, immunodepletion of DNA ligase IIIalpha did not significantly affect either pathway. Moreover, repair of normal abasic sites in wild-type and X-ray cross-complementing gene 1 (XRCC1)-DNA ligase IIIalpha-immunodepleted cell extracts involved similar proportions of short- and long-patch repair events. This suggests that DNA ligase I was able to efficiently substitute the XRCC1-DNA ligase IIIalpha complex during short-patch repair.     

Beyond base excision repair: an evolving picture of mitochondrial DNA repair

Mitochondria are highly specialised organelles required for key cellular processes including ATP production through cellular respiration and controlling cell death via apoptosis. Unlike other organelles, mitochondria contain their own DNA genome which encodes both protein and RNA required for cellular respiration. Each cell may contain hundreds to thousands of copies of the mitochondrial genome, which is essential for normal cellular function – deviation of mitochondrial DNA (mtDNA) copy number is associated with cellular ageing and disease. Furthermore, mtDNA lesions can arise from both endogenous or exogenous sources and must either be tolerated or corrected to preserve mitochondrial function. Importantly, replication of damaged mtDNA can lead to stalling and introduction of mutations or genetic loss, mitochondria have adapted mechanisms to repair damaged DNA. These mechanisms rely on nuclear-encoded DNA repair proteins that are translocated into the mitochondria.Despite the presence of many known nuclear DNA repair proteins being found in the mitochondrial proteome, it remains to be established which DNA repair mechanisms are functional in mammalian mitochondria. Here, we summarise the existing and emerging research, alongside examining proteomic evidence, demonstrating that mtDNA damage can be repaired using Base Excision Repair (BER), Homologous Recombination (HR) and Microhomology-mediated End Joining (MMEJ). Critically, these repair mechanisms do not operate in isolation and evidence for interplay between pathways and repair associated with replication is discussed. Importantly, characterising non-canonical functions of key proteins and understanding the bespoke pathways used to tolerate, repair or bypass DNA damage will be fundamental in fully understanding the causes of mitochondrial genome mutations and mitochondrial dysfunction.     

Inhibition of short patch and long patch base excision repair by an oxidized abasic site

5'-(2-Phosphoryl-1,4-dioxobutane) (DOB) is an oxidized abasic lesion that is produced by a variety of DNA damaging agents, including several antitumor antibiotics. DOB efficiently and irreversibly inhibits DNA polymerase β, an essential base excision repair enzyme in mammalian cells. The generality of this mode of inhibition by DOB is supported by the inactivation of DNA polymerase λ, which may serve as a possible backup for DNA polymerase β during abasic site repair. Protein digests suggest that Lys72 and Lys84, which are present in the lyase active site of DNA polymerase β, are modified by DOB. Monoaldehyde analogues of DOB substantiate the importance of the 1,4-dicarbonyl component of DOB for efficient inactivation of Pol β and the contribution of a freely diffusible electrophile liberated from the inhibitor by the enzyme. Inhibition of DNA polymerase β's lyase function is accompanied by inactivation of its DNA polymerase activity as well, which prevents long patch base excision repair of DOB. Overall, DOB is highly refractory to short patch and long patch base excision repair. Its recalcitrance to succumb to repair suggests that DOB is a significant source of the cytotoxicity of DNA damaging agents that produce it.     

Long patch base excision repair with purified human proteins. DNA ligase I as patch size mediator for DNA polymerases delta and epsilon

Among the different base excision repair pathways known, the long patch base excision repair of apurinic/apyrimidinic sites is an important mechanism that requires proliferating cell nuclear antigen. We have reconstituted this pathway using purified human proteins. Our data indicated that efficient repair is dependent on six components including AP endonuclease, replication factor C, proliferating cell nuclear antigen, DNA polymerases delta or epsilon, flap endonuclease 1, and DNA ligase I. Fine mapping of the nucleotide replacement events showed that repair patches extended up to a maximum of 10 nucleotides 3' to the lesion. However, almost 70% of the repair synthesis was confined to 2-4-nucleotide patches and DNA ligase I appeared to be responsible for limiting the repair patch length. Moreover, both proliferating cell nuclear antigen and flap endonuclease 1 are required for the production and ligation of long patch repair intermediates suggesting an important role of this complex in both excision and resynthesis steps.     

Substrate channeling in mammalian base excision repair pathways: passing the baton

The current model for base excision repair (BER) involves two general sub-pathways termed single-nucleotide BER and long patch BER that are distinguished by their repair patch sizes and the enzymes/co-factors involved. Both sub-pathways involve a series of sequential steps from initiation to completion of repair. The BER sub-pathways are designed to sequester the various intermediates, passing them along from one step to the next without allowing these toxic molecules to trigger cell cycle arrest, necrotic cell death, or apoptosis. Although a variety of DNA-protein and protein-protein interactions are known for the BER intermediates and enzymes/co-factors, the molecular mechanisms accounting for step-to-step coordination are not well understood. In the present study we designed an in vitro assay to explore the question of whether there is a channeling or "hand-off" of the repair intermediates during BER in vitro. The results show that when BER enzymes are pre-bound to the initial single-nucleotide BER intermediate, the DNA is channeled from apurinic/apyrimidinic endonuclease 1 to DNA polymerase β and then to DNA ligase. In the long patch BER subpathway, where the 5'-end of the incised strand is blocked, the intermediate after DNA polymerase β gap filling is not channeled to the subsequent enzyme, flap endonuclease 1. Instead, flap endonuclease 1 must recognize and bind to the intermediate in competition with other molecules.     

Lack of effect of hydroxyurea on base excision repair in mammalian cells

The effect of hydroxyurea on the initial steps of base excision repair has been examined in mammalian cells in 3 different proliferative states: i.e., quiescent cells, asynchronously growing cells undergoing multiple divisions prior to confluence; and synchronous cell populations undergoing the first cell cycle(s) after release from quiescence. Two parameters of the base excision repair pathway were examined: (1) The direct excision of 7-methylguanine from cellular DNA in the presence of increasing hydroxyurea concentrations was quantitated by high performance liquid chromatography; (2) the effects of hydroxyurea on the uracil DNA glycosylase were examined by quantitating the levels of this base excision repair enzyme in quiescent and proliferating cells. In quiescent cells, hydroxyurea at concentrations routinely used to quantitate DNA repair had no effect on the excision rates of 7-methylguanine examined over a span of 3 days; nor was there any effect on the specific activity of uracil DNA glycosylase in confluent cells. In asynchronously proliferating mammalian cells, identical hydroxyurea concentrations had no effect on the induction of the glycosylase. In synchronous growing cells HU had no effect on the temporal sequence of induction of uracil DNA glycosylase prior to DNA replication, nor on the extent of this induction. These results suggest that hydroxyurea at concentrations generally used to measure DNA repair has no effect on base excision repair.     

DNA synthesis and dRPase activities of polymerase beta are both essential for single-nucleotide patch base excision repair in mammalian cell extracts

In mammalian cells the majority of altered bases in DNA are processed through a single-nucleotide patch base excision repair mechanism. Base excision repair is initiated by a DNA glycosylase that removes a damaged base and generates an abasic site (AP site). This AP site is further processed by an AP endonuclease activity that incises the phosphodiester bond adjacent to the AP site and generates a strand break containing 3'-OH and 5'-sugar phosphate ends. In mammalian cells, the 5'-sugar phosphate is removed by the AP lyase activity of DNA polymerase beta (Pol beta). The same enzyme also fills the gap, and the DNA ends are finally rejoined by DNA ligase. We measured repair of oligonucleotide substrates containing a single AP site in cell extracts prepared from normal and Pol beta-null mouse cells and show that the reduced repair in Pol beta-null extracts can be complemented by addition of purified Pol beta. Using this complementation assay, we demonstrate that mutated Pol beta without dRPase activity is able to stimulate long patch BER. Mutant Pol beta deficient in DNA synthesis, but with normal dRPase activity, does not stimulate repair in Pol beta-null cells. However, under conditions where we measure base excision repair accomplished exclusively through a single-nucleotide patch BER, neither dRPase nor DNA synthesis mutants of Pol beta alone, or the two together, were able to complement the repair defect. These data suggest that the dRPase and DNA synthesis activities of Pol beta are coupled and that both of these Pol beta functions are essential during short patch BER and cannot be efficiently substituted by other cellular enzymes.     

A mechanism for the control of patch size in mammalian cell DNA excision repair

The hypothesis is suggested that size of the region excised in repair of UV-induced damage in mammalian cell is determined by the occurrence at random of a recognition sequence which terminates this excision process. The statistics of first occurrence times for a specific nucleotide sequence in a random chain are derived and shown to lead to an approximately random distribution of sizes around the average. The heterogeneity in sizes arising from a model are shown not to conflict with existing measurements. A sequence of length three or four is sufficient to account for the measured average size.     

Protection against methylation-induced cytotoxicity by DNA polymerase beta-dependent long patch base excision repair

Using a plasmid-based uracil-containing DNA substrate, we found that the long patch base excision repair (BER) activity of a wild-type mouse fibroblast extract was partially inhibited by an antibody to DNA polymerase beta (beta-pol). This suggests that beta-pol participates in long patch BER, in addition to single-nucleotide BER. In single-nucleotide BER, the deoxyribose phosphate (dRP) in the abasic site is removed by the lyase activity of beta-pol. Methoxyamine (MX) can react with the aldehyde of an abasic site, making it refractory to the beta-elimination step of the dRP lyase mechanism, thus blocking single-nucleotide BER. MX exposure sensitizes wild-type, but not beta-pol null mouse embryonic fibroblasts, to the cytotoxic effects of methyl methanesulfonate (MMS) and methylnitrosourea. Expression of beta-pol in the null cells restores the ability of MX to modulate sensitivity to MMS. The beta-pol null cells are known to be hypersensitive to MMS and methylnitrosourea, and in the presence of MX (i.e. under conditions where single-nucleotide BER is blocked) the null cells are still considerably more sensitive than wild-type. The data are consistent with a role of beta-pol in long patch BER, which helps protect cells against methylation damage-induced cytotoxicity.     

Molecular and biological roles of Ape1 protein in mammalian base excision repair

Many oxidative DNA lesions are handled well by base excision repair (BER), but some types may be problematic. Recent work indicates that 2-deoxyribonolactone (dL) is such a lesion by forming stable, covalent cross-links between the abasic residue and DNA repair proteins with lyase activity. In the case of DNA polymerase beta, the reaction is potentiated by incision of dL by Ape1, the major mammalian AP endonuclease. When repair is prevented, polymerase beta is the most reactive cross-linking protein in whole-cell extracts. Cross-linking with dL is largely avoided by processing the damage through the "long-patch" (multinucleotide) BER pathway. However, if excess damage leads to the accumulation of unrepaired oxidative lesions in DNA, there may be a danger of polymerase beta-mediated cross-link formation. Understanding how cells respond to such complex damage is an important issue. In addition to its role in defending against DNA damage caused by exogenous agents, Ape1 protein is essential for coping with the endogenous DNA damage in human cells grown in culture. Suppression of Ape1 using RNA-interference technology causes arrest of cell proliferation and activation of apoptosis in various cell types, correlated with the accumulation of unrepaired abasic DNA damage. Notably, all these effects are reversed by expression of the unrelated protein Apn1 of S. cerevisiae, which shares only the enzymatic repair function with Ape1 (AP endonuclease).     

Variation in base excision repair capacity

The major DNA repair pathway for coping with spontaneous forms of DNA damage, such as natural hydrolytic products or oxidative lesions, is base excision repair (BER). In particular, BER processes mutagenic and cytotoxic DNA lesions such as non-bulky base modifications, abasic sites, and a range of chemically distinct single-strand breaks. Defects in BER have been linked to cancer predisposition, neurodegenerative disorders, and immunodeficiency. Recent data indicate a large degree of sequence variability in DNA repair genes and several studies have associated BER gene polymorphisms with disease risk, including cancer of several sites. The intent of this review is to describe the range of BER capacity among individuals and the functional consequences of BER genetic variants. We also discuss studies that associate BER deficiency with disease risk and the current state of BER capacity measurement assays.