The Impact of SNP-Induced Amino Acid Substitutions L19P and G66R in the dRP-Lyase Domain of Human DNA Polymerase ß on Enzyme Activities.

Base excision repair (BER), which involves the sequential activity of DNA glycosylases, apurinic/apyrimidinic endonucleases, DNA polymerases, and DNA ligases, is one of the enzymatic systems that preserve the integrity of the genome. Normal BER is effective, but due to single-nucleotide polymorphisms (SNPs), the enzymes themselves-whose main function is to identify and eliminate damaged bases-can undergo amino acid changes. One of the enzymes in BER is DNA polymerase ß (Polß), whose function is to fill gaps in DNA. SNPs can significantly affect the catalytic activity of an enzyme by causing an amino acid substitution. In this work, pre-steady-state kinetic analyses and molecular dynamics simulations were used to examine the activity of naturally occurring variants of Polß that have the substitutions L19P and G66R in the dRP-lyase domain. Despite the substantial distance between the dRP-lyase domain and the nucleotidyltransferase active site, it was found that the capacity to form a complex with DNA and with an incoming dNTP is significantly altered by these substitutions. Therefore, the lower activity of the tested polymorphic variants may be associated with a greater number of unrepaired DNA lesions.

Human Polß Natural Polymorphic Variants G118V and R149I Affects Substate Binding and Catalysis.

DNA polymerase ß (Polß) expression is essential for the cell's response to DNA damage that occurs during natural cellular processes. Polß is considered the main reparative DNA polymerase, whose role is to fill the DNA gaps arising in the base excision repair pathway. Mutations in Polß can lead to cancer, neurodegenerative diseases, or premature aging. Many single-nucleotide polymorphisms have been identified in the POLB gene, but the consequences of these polymorphisms are not always clear. It is known that some polymorphic variants in the Polß sequence reduce the efficiency of DNA repair, thereby raising the frequency of mutations in the genome. In the current work, we studied two polymorphic variants (G118V and R149I separately) of human Polß that affect its DNA-binding region. It was found that each amino acid substitution alters Polß's affinity for gapped DNA. Each polymorphic variant also weakens its binding affinity for dATP. The G118V variant was found to greatly affect Polß's ability to fill gapped DNA and slowed the catalytic rate as compared to the wild-type enzyme. Thus, these polymorphic variants seem to decrease the ability of Polß to maintain base excision repair efficiency.

The Role of Natural Polymorphic Variants of DNA Polymerase ß in DNA Repair.

DNA polymerase ß (Polß) is considered the main repair DNA polymerase involved in the base excision repair (BER) pathway, which plays an important part in the repair of damaged DNA bases usually resulting from alkylation or oxidation. In general, BER involves consecutive actions of DNA glycosylases, AP endonucleases, DNA polymerases, and DNA ligases. It is known that protein-protein interactions of Polß with enzymes from the BER pathway increase the efficiency of damaged base repair in DNA. However natural single-nucleotide polymorphisms can lead to a substitution of functionally significant amino acid residues and therefore affect the catalytic activity of the enzyme and the accuracy of Polß action. Up-to-date databases contain information about more than 8000 SNPs in the gene of Polß. This review summarizes data on the in silico prediction of the effects of Polß SNPs on DNA repair efficacy; available data on cancers associated with SNPs of Polß; and experimentally tested variants of Polß. Analysis of the literature indicates that amino acid substitutions could be important for the maintenance of the native structure of Polß and contacts with DNA; others affect the catalytic activity of the enzyme or play a part in the precise and correct attachment of the required nucleotide triphosphate. Moreover, the amino acid substitutions in Polß can disturb interactions with enzymes involved in BER, while the enzymatic activity of the polymorphic variant may not differ significantly from that of the wild-type enzyme. Therefore, investigation regarding the effect of Polß natural variants occurring in the human population on enzymatic activity and protein-protein interactions is an urgent scientific task.

Isoforms of Base Excision Repair Enzymes Produced by Alternative Splicing.

Transcripts of many enzymes involved in base excision repair (BER) undergo extensive alternative splicing, but functions of the corresponding alternative splice variants remain largely unexplored. In this review, we cover the studies describing the common alternatively spliced isoforms and disease-associated variants of DNA glycosylases, AP-endonuclease 1, and DNA polymerase beta. We also discuss the roles of alternative splicing in the regulation of their expression, catalytic activities, and intracellular transport.

SNP-Associated Substitutions of Amino Acid Residues in the dNTP Selection Subdomain Decrease Polß Polymerase Activity.

In the cell, DNA polymerase ß (Polß) is involved in many processes aimed at maintaining genome stability and is considered the main repair DNA polymerase participating in base excision repair (BER). Polß can fill DNA gaps formed by other DNA repair enzymes. Single-nucleotide polymorphisms (SNPs) in the POLB gene can affect the enzymatic properties of the resulting protein, owing to possible amino acid substitutions. For many SNP-associated Polß variants, an association with cancer, owing to changes in polymerase activity and fidelity, has been shown. In this work, kinetic analyses and molecular dynamics simulations were used to examine the activity of naturally occurring polymorphic variants G274R, G290C, and R333W. Previously, the amino acid substitutions at these positions have been found in various types of tumors, implying a specific role of Gly-274, Gly-290, and Arg-333 in Polß functioning. All three polymorphic variants had reduced polymerase activity. Two substitutions-G274R and R333W-led to the almost complete disappearance of gap-filling and primer elongation activities, a decrease in the deoxynucleotide triphosphate-binding ability, and a lower polymerization constant, due to alterations of local contacts near the replaced amino acid residues. Thus, variants G274R, G290C, and R333W may be implicated in an elevated level of unrepaired DNA damage.

Oncometabolite 2-hydroxyglutarate suppresses basal protein levels of DNA polymerase beta that enhances alkylating agent and PARG inhibition induced cytotoxicity.

Mutations in isocitrate dehydrogenase isoform 1 (IDH1) are primarily found in secondary glioblastoma (GBM) and low-grade glioma but are rare in primary GBM. The standard treatment for GBM includes radiation combined with temozolomide, an alkylating agent. Fortunately, IDH1 mutant gliomas are sensitive to this treatment, resulting in a more favorable prognosis. However, it's estimated that up to 75 % of IDH1 mutant gliomas will progress to WHO grade IV over time and develop resistance to alkylating agents. Therefore, understanding the mechanism(s) by which IDH1 mutant gliomas confer sensitivity to alkylating agents is crucial for developing targeted chemotherapeutic approaches. The base excision repair (BER) pathway is responsible for repairing most base damage induced by alkylating agents. Defects in this pathway can lead to hypersensitivity to these agents due to unresolved DNA damage. The coordinated assembly and disassembly of BER protein complexes are essential for cell survival and for maintaining genomic integrity following alkylating agent exposure. These complexes rely on poly-ADP-ribose formation, an NAD+-dependent post-translational modification synthesized by PARP1 and PARP2 during the BER process. At the lesion site, poly-ADP-ribose facilitates the recruitment of XRCC1. This scaffold protein helps assemble BER proteins like DNA polymerase beta (Polß), a bifunctional DNA polymerase containing both DNA synthesis and 5'-deoxyribose-phosphate lyase (5'dRP lyase) activity. Here, we confirm that IDH1 mutant glioma cells have defective NAD+ metabolism, but still produce sufficient nuclear NAD+ for robust PARP1 activation and BER complex formation in response to DNA damage. However, the overproduction of 2-hydroxyglutarate, an oncometabolite produced by the IDH1 R132H mutant protein, suppresses BER capacity by reducing Polß protein levels. This defines a novel mechanism by which the IDH1 mutation in gliomas confers cellular sensitivity to alkylating agents and to inhibitors of the poly-ADP-ribose glycohydrolase, PARG.

Mouse models to explore the biological and organismic role of DNA polymerase beta.

Gene knock-out (KO) mouse models for DNA polymerase beta (Polß) revealed that loss of Polß leads to neonatal lethality, highlighting the critical organismic role for this DNA polymerase. While biochemical analysis and gene KO cell lines have confirmed its biochemical role in base excision repair and in TET-mediated demethylation, more long-lived mouse models continue to be developed to further define its organismic role. The Polb-KO mouse was the first of the Cre-mediated tissue-specific KO mouse models. This technology was exploited to investigate roles for Polß in V(D)J recombination (variable-diversity-joining rearrangement), DNA demethylation, gene complementation, SPO11-induced DNA double-strand break repair, germ cell genome stability, as well as neuronal differentiation, susceptibility to genotoxin-induced DNA damage, and cancer onset. The revolution in knock-in (KI) mouse models was made possible by CRISPR/cas9-mediated gene editing directly in C57BL/6 zygotes. This technology has helped identify phenotypes associated with germline or somatic mutants of Polß. Such KI mouse models have helped uncover the importance of key Polß active site residues or specific Polß enzyme activities, such as the PolbY265C mouse that develops lupus symptoms. More recently, we have used this KI technology to mutate the Polb gene with two codon changes, yielding the PolbL301R/V303R mouse. In this KI mouse model, the expressed Polß protein cannot bind to its obligate heterodimer partner, Xrcc1. Although the expressed mutant Polß protein is proteolytically unstable and defective in recruitment to sites of DNA damage, the homozygous PolbL301R/V303R mouse is viable and fertile, yet small in stature. We expect that this and additional targeted mouse models under development are poised to reveal new biological and organismic roles for Polß.

Cellular roles of DNA polymerase beta.

Since its discovery and purification in 1971, DNA polymerase ß (Pol ß) is one of the most well-studied DNA polymerases. Pol ß is a key enzyme in the base excision repair (BER) pathway that functions in gap filling DNA synthesis subsequent to the excision of damaged DNA bases. A major focus of our studies is on the cellular roles of Pol ß. We have shown that germline and tumor-associated variants of Pol ß catalyze aberrant BER that leads to genomic instability and cellular transformation. Our studies suggest that Pol ß is critical for the maintenance of genomic stability and that it is a tumor suppressor. We have also shown that Pol ß functions during Prophase I of meiosis. Pol ß localizes to the synaptonemal complex and is critical for removal of the Spo11 complex from the 5' ends of double-strand breaks. Studies with Pol ß mutant mice are currently being undertaken to more clearly understand the function of Pol ß during meiosis. In this review, we will highlight our contributions from our studies of Pol ß germline and cancer-associated variants.

The Activity of Natural Polymorphic Variants of Human DNA Polymerase ß Having an Amino Acid Substitution in the Transferase Domain.

To maintain the integrity of the genome, there is a set of enzymatic systems, one of which is base excision repair (BER), which includes sequential action of DNA glycosylases, apurinic/apyrimidinic endonucleases, DNA polymerases, and DNA ligases. Normally, BER works efficiently, but the enzymes themselves (whose primary function is the recognition and removal of damaged bases) are subject to amino acid substitutions owing to natural single-nucleotide polymorphisms (SNPs). One of the enzymes in BER is DNA polymerase ß (Polß), whose function is to fill gaps in DNA with complementary dNMPs. It is known that many SNPs can cause an amino acid substitution in this enzyme and a significant decrease in the enzymatic activity. In this study, the activity of four natural variants of Polß, containing substitution E154A, G189D, M236T, or R254I in the transferase domain, was analyzed using molecular dynamics simulations and pre-steady-state kinetic analyses. It was shown that all tested substitutions lead to a significant reduction in the ability to form a complex with DNA and with incoming dNTP. The G189D substitution also diminished Polß catalytic activity. Thus, a decrease in the activity of studied mutant forms may be associated with an increased risk of damage to the genome.

Polß/XRCC1 heterodimerization dictates DNA damage recognition and basal Polß protein levels without interfering with mouse viability or fertility.

DNA Polymerase ß (Polß) performs two critical enzymatic steps during base excision repair (BER) - gap filling (nucleotidyl transferase activity) and gap tailoring (dRP lyase activity). X-ray repair cross complementing 1 (XRCC1) facilitates the recruitment of Polß to sites of DNA damage through an evolutionarily conserved Polß/XRCC1 interaction interface, the V303 loop. While previous work describes the importance of the Polß/XRCC1 interaction for human Polß protein stability and recruitment to sites of DNA damage, the impact of disrupting the Polß/XRCC1 interface on animal viability, physiology, and fertility is unknown. Here, we characterized the effect of disrupting Polß/XRCC1 heterodimerization in mice and mouse cells by complimentary approaches. First, we demonstrate, via laser micro-irradiation, that mouse Polß amino acid residues L301 and V303 are critical to facilitating Polß recruitment to sites of DNA damage. Next, we solved the crystal structures of mouse wild type Polß and a mutant protein harboring alterations in residues L301 and V303 (L301R/V303R). Our structural analyses suggest that Polß amino acid residue V303 plays a role in maintaining an interaction with the oxidized form of XRCC1. Finally, we created CRISPR/Cas9-modified Polb mice with homozygous L301R/V303R mutations (PolbL301R-V303R/L301R-V303R) that are fertile yet exhibit 15% reduced body weight at 17 weeks of age, as compared to heterozygous mice. Fibroblasts derived from PolbL301R-V303R/L301R-V303R mice demonstrate that mutation of mouse Polß's XRCC1 interaction domain leads to an ∼85% decrease in Polß protein levels. In all, these studies are consistent with a role for the oxidized form of XRCC1 in providing stability to the Polß protein through Polß/XRCC1 heterodimer formation.

Defective DNA polymerase beta invoke a cytosolic DNA mediated inflammatory response.

Base excision repair (BER) has evolved to maintain the genomic integrity of DNA following endogenous and exogenous agent induced DNA base damage. In contrast, aberrant BER induces genomic instability, promotes malignant transformation and can even trigger cancer development. Previously, we have shown that deoxyribo-5'-phosphate (dRP) lyase deficient DNA polymerase beta (POLB) causes replication associated genomic instability and sensitivity to both endogenous and exogenous DNA damaging agents. Specifically, it has been established that this loss of dRP lyase function promotes inflammation associated gastric cancer. However, the way that aberrant POLB impacts the immune signaling and inflammatory responses is still unknown. Here we show that a dRP lyase deficient variant of POLB (Leu22Pro, or L22P) increases mitotic dysfunction associated genomic instability, which eventually leads to a cytosolic DNA mediated inflammatory response. Furthermore, poly(ADP-ribose) polymerase 1 inhibition exacerbates chromosomal instability and enhances the cytosolic DNA mediated inflammatory response. Our results suggest that POLB plays a significant role in modulating inflammatory signaling, and they provide a mechanistic basis for future potential cancer immunotherapies.

Molecular and Functional Characteristics of DNA Polymerase Beta-Like Enzymes From Trypanosomatids.

Trypanosomatids are a group of primitive unicellular eukaryotes that can cause diseases in plants, insects, animals, and humans. Kinetoplast genome integrity is key to trypanosomatid cell survival and viability. Kinetoplast DNA (kDNA) is usually under attack by reactive oxygen and nitric species (ROS and RNS), damaging the DNA, and the cells must remove and repair those oxidatively generated lesions in order to survive and proliferate. Base excision repair (BER) is a well-conserved pathway for DNA repair after base damage, single-base loss, and single-strand breaks, which can arise from ROS, RSN, environmental genotoxic agents, and UV irradiation. A powerful BER system has been described in the T. cruzi kinetoplast and it is mainly carried out by DNA polymerase ß (pol ß) and DNA polymerase ß-PAK (pol ß-PAK), which are kinetoplast-located in T. cruzi as well as in other trypanosomatids. Both pol ß and pol ß-PAK belong to the X-family of DNA polymerases (pol X family), perform BER in trypanosomatids, and display intrinsic 5-deoxyribose phosphate (dRP) lyase and DNA polymerase activities. However, only Pol ß-PAK is able to carry out trans-lesion synthesis (TLS) across 8oxoG lesions. T. cruzi cells overexpressing pol ß are more resistant to ROS and are also more efficient to repair 8oxoG compared to control cells. Pol ß seems to play a role in kDNA replication, since it associates with kinetoplast antipodal sites in those development stages in trypanosomatids which are competent for cell replication. ROS treatment of cells induces the overexpression of pol ß, indicating that plays a role in kDNA repair. In this review, we will summarize the main features of trypanosomatid minicircle kDNA replication and the biochemical characteristics of pol ß-like enzymes and their involvement in BER and kDNA replication. We also summarize key structural features of trypanosomatid pol ß compared to their mammalian (human) counterpart.

DNA glycosylase deficiency leads to decreased severity of lupus in the Polb-Y265C mouse model.

The Polb gene encodes DNA polymerase beta (Pol ß), a DNA polymerase that functions in base excision repair (BER) and microhomology-mediated end-joining. The Pol ß-Y265C protein exhibits low catalytic activity and fidelity, and is also deficient in microhomology-mediated end-joining. We have previously shown that the PolbY265C/+ and PolbY265C/C mice develop lupus. These mice exhibit high levels of antinuclear antibodies and severe glomerulonephritis. We also demonstrated that the low catalytic activity of the Pol ß-Y265C protein resulted in accumulation of BER intermediates that lead to cell death. Debris released from dying cells in our mice could drive development of lupus. We hypothesized that deletion of the Neil1 and Ogg1 DNA glycosylases that act upstream of Pol ß during BER would result in accumulation of fewer BER intermediates, resulting in less severe lupus. We found that high levels of antinuclear antibodies are present in the sera of PolbY265C/+ mice deleted of Ogg1 and Neil1 DNA glycosylases. However, these mice develop significantly less severe renal disease, most likely due to high levels of IgM in their sera.

Predicting the Role of DNA Polymerase ß Alone or with KRAS Mutations in Advanced NSCLC Patients Receiving Platinum-Based Chemotherapy.

Clinical data suggest that only a subgroup of non-small cell lung cancer (NSCLC) patients has long-term benefits after front-line platinum-based therapy. We prospectively investigate whether KRAS status and DNA polymerase ß expression could help identify patients responding to platinum compounds. Prospectively enrolled, advanced NSCLC patients treated with a first-line regimen containing platinum were genotyped for KRAS and centrally evaluated for DNA polymerase ß expression. Overall survival (OS), progression-free survival (PFS), and the objective response rate (ORR) were recorded. Patients with KRAS mutations had worse OS (hazard ratio (HR): 1.37, 95% confidence interval (95% CI): 0.70-2.27). Negative DNA polymerase ß staining identified a subgroup with worse OS than patients expressing the protein (HR: 1.43, 95% CI: 0.57-3.57). The addition of KRAS to the analyses further worsened the prognosis of patients with negative DNA polymerase ß staining (HR: 1.67, 95% CI: 0.52-5.56). DNA polymerase ß did not influence PFS and ORR. KRAS may have a negative role in platinum-based therapy responses in NSCLC, but its impact is limited. DNA polymerase ß, when not expressed, might indicate a group of patients with poor outcomes. KRAS mutations in tumors not expressing DNA polymerase ß further worsens survival. Therefore, these two biomarkers together might well identify patients for whom alternatives to platinum-based chemotherapy should be used.

Mutation in DNA Polymerase Beta Causes Spontaneous Chromosomal Instability and Inflammation-Associated Carcinogenesis in Mice.

DNA polymerase beta (Pol ß) is a key enzyme in the base excision repair (BER) pathway. Pol ß is mutated in approximately 40% of human tumors in small-scale studies. The 5´-deoxyribose-5-phosphate (dRP) lyase domain of Pol ß is responsible for DNA end tailoring to remove the 5' phosphate group. We previously reported that the dRP lyase activity of Pol ß is critical to maintain DNA replication fork stability and prevent cellular transformation. In this study, we tested the hypothesis that the human gastric cancer associated variant of Pol ß (L22P) has the ability to promote spontaneous chromosomal instability and carcinogenesis in mice. We constructed a Pol ß L22P conditional knock-in mouse model and found that L22P enhances hyperproliferation and DNA double strand breaks (DSBs) in stomach cells. Moreover, mouse embryonic fibroblasts (MEFs) derived from L22P mice frequently induce abnormal numbers of chromosomes and centrosome amplification, leading to chromosome segregation errors. Importantly, L22P mice exhibit chronic inflammation accompanied by stomach tumors. These data demonstrate that the human cancer-associated variant of Pol ß can contribute to chromosomal instability and cancer development.

Aberrant DNA Polymerase Beta Enhances H. pylori Infection Induced Genomic Instability and Gastric Carcinogenesis in Mice.

H. pylori is a significant risk factor of gastric cancer that induces chronic inflammation and oxidative DNA damage to promote gastric carcinoma. Base excision repair (BER) is required to maintain the genome integrity and prevent oxidative DNA damage. Mutation in DNA polymerase beta (Pol ß) impacts BER efficiency and has been reported in approximately 30-40% of gastric carcinoma tumors. In this study, we examined whether reduced BER capacity associated with mutation in the POLB gene, along with increased DNA damage generated by H. pylori infection, accelerates gastric cancer development. By infecting a Pol ß mutant mouse model that lacks dRP lyase with H. pylori, we show that reactive oxygen and nitrogen species (RONS) mediated DNA damage is accumulated in Pol ß mutant mice (L22P). In addition, H. pylori infection in Leu22Pro (L22P) mice significantly increases inducible nitric oxide synthesis (iNOS) mediated chronic inflammation. Our data show that L22P mice exhibited accelerated H. pylori induced carcinogenesis and increased tumor incidence. This work shows that Pol ß mediated DNA repair under chronic inflammation conditions is an important suppressor of H. pylori induced stomach carcinogenesis.

Loss of DNA polymerase ß induces cellular senescence.

We aim to establish that accelerated aging and premature cellular senescence seen in individuals with Down syndrome is related to reduced DNA polymeraseß. We report here that primary fibroblasts from Down syndrome individuals exhibit greater SA-ß-gal staining (fourfold increase, P < 0.001), increased p16 transcript abundance (threefold increase, P < 0.01), and reduced HMGB1 nuclear localization (1.5-fold lower, P < 0.01). We also find that DNA polymerase ß expression is significantly reduced in Down syndrome primary fibroblasts (53% decline, P < 0.01). To evaluate whether DNA polymerase ß might be causative in senescence induction, we evaluated the impact of murine DNA polymerase ß nullizygosity on senescence. We find that unexposed DNA polymerase ß -null primary fibroblasts exhibit a robust increase in the number of senescent cells compared to wild-type (11-fold, P < 0.001), demonstrating that loss DNA polymerase ß is sufficient to induce senescence. We also see an additional increase in response to hydroxyurea (threefold greater than WT-HU, P < 0.05). These data demonstrate that loss of DNA polymerase ß is sufficient to induce senescence. Additionally, we report a significant induction in spontaneous DNA double strand breaks in DNA polymerase ß null MEFs (fivefold increase from wild-type, P < 0.0001). Our findings strongly suggest that DNA polymerase ß is causative in senescence induction, reasonably pointing to DNA polymerase ß as a likely factor driving the premature senescence in Down syndrome. Environ. Mol. Mutagen. 59:603-612, 2018. © 2018 Wiley Periodicals, Inc.

Molecular dynamics simulations suggest changes in electrostatic interactions as a potential mechanism through which serine phosphorylation inhibits DNA Polymerase ß's activity.

DNA polymerase ß is a 39kDa enzyme that is a major component of Base Excision Repair in human cells. The enzyme comprises two major domains, a 31kDa domain responsible for the polymerase activity and an 8kDa domain, which bind ssDNA and has a deoxyribose phosphate (dRP) lyase activity. DNA polymerase ß was shown to be phosphorylated in vitro with protein kinase C (PKC) at serines 44 and 55 (S44 and S55), resulting in loss of its polymerase enzymic activity, but not its ability to bind ssDNA. In this study, we investigate the potential phosphorylation-induced structural changes for DNA polymerase ß using molecular dynamics. The simulations show drastic conformational changes of the polymerase structure as a result of S44 phosphorylation. Phosphorylation-induced conformational changes transform the closed (active) enzyme structure into an open one. Further analysis of the results points to a key hydrogen bond and newly formed salt bridges as potential drivers of these structural fluctuations. The changes observed with S44/55 and S55 phosphorylation were less dramatic than S44 and the integrity of the H-bond was not compromised. Thus the phosphorylation of S44 is likely the major contributor to structural fluctuations that lead to loss of enzymatic activity.

Risky repair: DNA-protein crosslinks formed by mitochondrial base excision DNA repair enzymes acting on free radical lesions.

Oxygen is both necessary and dangerous for aerobic cell function. ATP is most efficiently made by the electron transport chain, which requires oxygen as an electron acceptor. However, the presence of oxygen, and to some extent the respiratory chain itself, poses a danger to cellular components. Mitochondria, the sites of oxidative phosphorylation, have defense and repair pathways to cope with oxidative damage. For mitochondrial DNA, an essential pathway is base excision repair, which acts on a variety of small lesions. There are instances, however, in which attempted DNA repair results in more damage, such as the formation of a DNA-protein crosslink trapping the repair enzyme on the DNA. That is the case for mitochondrial DNA polymerase γ acting on abasic sites oxidized at the 1-carbon of 2-deoxyribose. Such DNA-protein crosslinks presumably must be removed in order to restore function. In nuclear DNA, ubiquitylation of the crosslinked protein and digestion by the proteasome are essential first processing steps. How and whether such mechanisms operate on DNA-protein crosslinks in mitochondria remains to be seen.

DNA Polymerase Beta Participates in Mitochondrial DNA Repair.

We have detected DNA polymerase beta (Polß), known as a key nuclear base excision repair (BER) protein, in mitochondrial protein extracts derived from mammalian tissue and cells. Manipulation of the N-terminal sequence affected the amount of Polß in the mitochondria. Using Polß fragments, mitochondrion-specific protein partners were identified, with the interactors functioning mainly in DNA maintenance and mitochondrial import. Of particular interest was the identification of the proteins TWINKLE, SSBP1, and TFAM, all of which are mitochondrion-specific DNA effectors and are known to function in the nucleoid. Polß directly interacted functionally with the mitochondrial helicase TWINKLE. Human kidney cells with Polß knockout (KO) had higher endogenous mitochondrial DNA (mtDNA) damage. Mitochondrial extracts derived from heterozygous Polß mouse tissue and KO cells had lower nucleotide incorporation activity. Mouse-derived Polß null fibroblasts had severely affected metabolic parameters. Indeed, gene knockout of Polß caused mitochondrial dysfunction, including reduced membrane potential and mitochondrial content. We show that Polß is a mitochondrial polymerase involved in mtDNA maintenance and is required for mitochondrial homeostasis.