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.

Mitochondrial transcription factor A (TFAM) has 5'-deoxyribose phosphate lyase activity in vitro.

Mitochondrial DNA (mtDNA) plays a key role in mitochondrial and cellular functions. mtDNA is maintained by active DNA turnover and base excision repair (BER). In BER, one of the toxic repair intermediates is 5'-deoxyribose phosphate (5'dRp). Human mitochondrial DNA polymerase γ has weak dRp lyase activities, and another known dRp lyase in the nucleus, human DNA polymerase ß, can also localize to mitochondria in certain cell and tissue types. Nonetheless, whether additional proteins have the ability to remove 5'dRp in mitochondria remains unknown. Our prior work on the AP lyase activity of mitochondrial transcription factor A (TFAM) has prompted us to examine its ability to remove 5'dRp residues in vitro. TFAM is the primary DNA-packaging factor in human mitochondria and interacts with mitochondrial DNA extensively. Our data demonstrate that TFAM has the dRp lyase activity with different DNA substrates. Under single-turnover conditions, TFAM removes 5'dRp residues at a rate comparable to that of DNA polymerase (pol) ß, albeit slower than that of pol λ. Among the three proteins examined, pol λ shows the highest single-turnover rates in dRp lyase reactions. The catalytic effect of TFAM is facilitated by lysine residues of TFAM via Schiff base chemistry, as evidenced by the observation of dRp-lysine adducts in mass spectrometry experiments. The catalytic effect of TFAM observed here is analogous to the AP lyase activity of TFAM reported previously. Together, these results suggest a potential role of TFAM in preventing the accumulation of toxic DNA repair intermediates.

DNA polymerase λ Loop1 variant yields unexpected gain-of-function capabilities in nonhomologous end-joining.

DNA polymerases lambda (Polλ) and mu (Polµ) are X-Family polymerases that participate in DNA double-strand break (DSB) repair by the nonhomologous end-joining pathway (NHEJ). Both polymerases direct synthesis from one DSB end, using template derived from a second DSB end. In this way, they promote the NHEJ ligation step and minimize the sequence loss normally associated with this pathway. The two polymerases differ in cognate substrate, as Polλ is preferred when synthesis must be primed from a base-paired DSB end, while Polµ is required when synthesis must be primed from an unpaired DSB end. We generated a Polλ variant (PolλKGET) that retained canonical Polλ activity on a paired end-albeit with reduced incorporation fidelity. We recently discovered that the variant had unexpectedly acquired the activity previously unique to Polµ-synthesis from an unpaired primer terminus. Though the sidechains of the Loop1 region make no contact with the DNA substrate, PolλKGET Loop1 amino acid sequence is surprisingly essential for its unique activity during NHEJ. Taken together, these results underscore that the Loop1 region plays distinct roles in different Family X polymerases.

Unfilled gaps by polß lead to aberrant ligation by LIG1 at the downstream steps of base excision repair pathway.

Base excision repair (BER) involves the tightly coordinated function of DNA polymerase ß (polß) and DNA ligase I (LIG1) at the downstream steps. Our previous studies emphasize that defective substrate-product channeling, from gap filling by polß to nick sealing by LIG1, can lead to interruptions in repair pathway coordination. Yet, the molecular determinants that dictate accurate BER remains largely unknown. Here, we demonstrate that a lack of gap filling by polß leads to faulty repair events and the formation of deleterious DNA intermediates. We dissect how ribonucleotide challenge and cancer-associated mutations could adversely impact the ability of polß to efficiently fill the one nucleotide gap repair intermediate which subsequently results in gap ligation by LIG1, leading to the formation of single-nucleotide deletion products. Moreover, we demonstrate that LIG1 is not capable of discriminating against nick DNA containing a 3'-ribonucleotide, regardless of base-pairing potential or damage. Finally, AP-Endonuclease 1 (APE1) shows distinct substrate specificity for the exonuclease removal of 3'-mismatched bases and ribonucleotides from nick repair intermediate. Overall, our results reveal that unfilled gaps result in impaired coordination between polß and LIG1, defining a possible type of mutagenic event at the downstream steps where APE1 could provide a proofreading role to maintain BER efficiency.

DNA polymerase beta connects tumorigenicity with the circadian clock in liver cancer through the epigenetic demethylation of Per1.

The circadian-controlled DNA repair exhibits a strong diurnal rhythm. Disruption in circadian clock and DNA repair is closely linked with hepatocellular carcinoma (HCC) progression, but the mechanism remains unknown. Here, we show that polymerase beta (POLB), a critical enzyme in the DNA base excision repair pathway, is rhythmically expressed at the translational level in mouse livers. Hepatic POLB dysfunction dampens clock homeostasis, whereas retards HCC progression, by mediating the methylation of the 4th CpG island on the 5'UTR of clock gene Per1. Clinically, POLB is overexpressed in human HCC samples and positively associated with poor prognosis. Furthermore, the hepatic rhythmicity of POLB protein expression is orchestrated by Calreticulin (CALR). Our findings provide important insights into the molecular mechanism underlying the synergy between clock and food signals on the POLB-driven BER system and reveal new clock-dependent carcinogenetic effects of POLB. Therefore, chronobiological modulation of POLB may help to promote precise interventions for HCC.

Coordination between human DNA polymerase ß and apurinic/apyrimidinic endonuclease 1 in the course of DNA repair.

Coordination of enzymatic activities in the course of base excision repair (BER) is essential to ensure complete repair of damaged bases. Two major mechanisms underlying the coordination of BER are known today: the "passing the baton" model and a model of preassembled stable multiprotein repair complexes called "repairosomes." In this work, we aimed to elucidate the coordination between human apurinic/apyrimidinic (AP) endonuclease APE1 and DNA polymerase Polß in BER through studying an impact of APE1 on Polß-catalyzed nucleotide incorporation into different model substrates that mimic different single-strand break (SSB) intermediates arising along the BER pathway. It was found that APE1's impact on separate stages of Polß's catalysis depends on the nature of a DNA substrate. In this complex, APE1 removed 3' blocking groups and corrected Polß-catalyzed DNA synthesis in a coordinated manner. Our findings support the hypothesis that Polß not only can displace APE1 from damaged DNA within the "passing the baton" model but also performs the gap-filling reaction in the ternary complex with APE1 according to the "repairosome" model. Taken together, our results provide new insights into coordination between APE1 and Polß during the BER process.

Non-Catalytic Domains of DNA Polymerase λ: Influence on Enzyme Activity and Its Regulation.

DNA polymerase λ (Polλ) belongs to the same structural X-family as DNA polymerase ß, the main polymerase of base excision repair. The role of Polλ in this process remains not fully understood. A significant difference between the two DNA polymerases is the presence of an extended non-catalytic N-terminal region in the Polλ structure. The influence of this region on the interaction of Polλ with DNA and multifunctional proteins, poly(ADP-ribose)polymerase 1 (PARP1) and replication protein A (RPA), was studied in detail for the first time. The data obtained suggest that non-catalytic Polλ domains play a suppressor role both in relation to the polymerase activity of the enzyme and in interaction with DNA and PARP1.

Impeding DNA Polymerase ß Activity by Oleic Acid to Inhibit Base Excision Repair and Induce Mitochondrial Dysfunction in Hepatic Cells.

Free fatty acids (FFAs) hepatic accumulation and the resulting oxidative stress contribute to several chronic liver diseases including nonalcoholic steatohepatitis. However, the underlying pathological mechanisms remain unclear. In this study, we propose a novel mechanism whereby the toxicity of FFAs detrimentally affects DNA repair activity. Specifically, we have discovered that oleic acid (OA), a prominent dietary free fatty acid, inhibits the activity of DNA polymerase ß (Pol ß), a crucial enzyme involved in base excision repair (BER), by actively competing with 2'-deoxycytidine-5'-triphosphate. Consequently, OA hinders the efficiency of BER, leading to the accumulation of DNA damage in hepatocytes overloaded with FFAs. Additionally, the excessive presence of both OA and palmitic acid (PA) lead to mitochondrial dysfunction in hepatocytes. These findings suggest that the accumulation of FFAs hampers Pol ß activity and contributes to mitochondrial dysfunction, shedding light on potential pathogenic mechanisms underlying FFAs-related diseases.

DinB (DNA polymerase IV), ImuBC and RpoS contribute to the generation of ciprofloxacin-resistance mutations in Pseudomonas aeruginosa.

We investigated the role(s) of the damage-inducible SOS response dinB and imuBC gene products in the generation of ciprofloxacin-resistance mutations in the important human opportunistic bacterial pathogen, Pseudomonas aeruginosa. We found that the overall numbers of ciprofloxacin resistant (CipR) mutants able to be recovered under conditions of selection were significantly reduced when the bacterial cells concerned carried a defective dinB gene, but could be elevated to levels approaching wild-type when these cells were supplied with the dinB gene on a plasmid vector; in turn, firmly establishing a role for the dinB gene product, error-prone DNA polymerase IV, in the generation of CipR mutations in P. aeruginosa. Further, we report that products of the SOS-regulated imuABC gene cassette of this organism, ImuB and the error-prone ImuC DNA polymerase, are also involved in generating CipR mutations in this organism, since the yields of CipR mutations were substantially decreased in imuB- or imuC-defective cells compared to wild-type. Intriguingly, we found that the mutability of a dinB-defective strain could not be rescued by overexpression of the imuBC genes. And similarly, overexpression of the dinB gene either only modestly or else failed to restore CipR mutations in imuB- or imuC-defective cells, respectively. Combined, these results indicated that the products of the dinB and imuBC genes were acting in the same pathway leading to the generation of CipR mutations in P. aeruginosa. In addition, we provide evidence indicating that the general stress response sigma factor σs, RpoS, is required for mutagenesis in this organism and is in part at least modulating the dinB (DNA polymerase IV)-dependent mutational process. Altogether, these data provide further insight into the complexity and multifaceted control of the mutational mechanism(s) contributing to the generation of ciprofloxacin-resistance mutations in P. aeruginosa.

The Impact of Human DNA Glycosylases on the Activity of DNA Polymerase ß toward Various Base Excision Repair Intermediates.

Base excision repair (BER) is one of the important systems for the maintenance of genome stability via repair of DNA lesions. BER is a multistep process involving a number of enzymes, including damage-specific DNA glycosylases, apurinic/apyrimidinic (AP) endonuclease 1, DNA polymerase ß, and DNA ligase. Coordination of BER is implemented by multiple protein-protein interactions between BER participants. Nonetheless, mechanisms of these interactions and their roles in the BER coordination are poorly understood. Here, we report a study on Polß's nucleotidyl transferase activity toward different DNA substrates (that mimic DNA intermediates arising during BER) in the presence of various DNA glycosylases (AAG, OGG1, NTHL1, MBD4, UNG, or SMUG1) using rapid-quench-flow and stopped-flow fluorescence approaches. It was shown that Polß efficiently adds a single nucleotide into different types of single-strand breaks either with or without a 5'-dRP-mimicking group. The obtained data indicate that DNA glycosylases AAG, OGG1, NTHL1, MBD4, UNG, and SMUG1, but not NEIL1, enhance Polß's activity toward the model DNA intermediates.

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.

Abasic site-peptide cross-links are blocking lesions repaired by AP endonucleases.

Apurinic/apyrimidinic (AP) sites are abundant DNA lesions arising from spontaneous hydrolysis of the N-glycosidic bond and as base excision repair (BER) intermediates. AP sites and their derivatives readily trap DNA-bound proteins, resulting in DNA-protein cross-links. Those are subject to proteolysis but the fate of the resulting AP-peptide cross-links (APPXLs) is unclear. Here, we report two in vitro models of APPXLs synthesized by cross-linking of DNA glycosylases Fpg and OGG1 to DNA followed by trypsinolysis. The reaction with Fpg produces a 10-mer peptide cross-linked through its N-terminus, while OGG1 yields a 23-mer peptide attached through an internal lysine. Both adducts strongly blocked Klenow fragment, phage RB69 polymerase, Saccharolobus solfataricus Dpo4, and African swine fever virus PolX. In the residual lesion bypass, mostly dAMP and dGMP were incorporated by Klenow and RB69 polymerases, while Dpo4 and PolX used primer/template misalignment. Of AP endonucleases involved in BER, Escherichia coli endonuclease IV and its yeast homolog Apn1p efficiently hydrolyzed both adducts. In contrast, E. coli exonuclease III and human APE1 showed little activity on APPXL substrates. Our data suggest that APPXLs produced by proteolysis of AP site-trapped proteins may be removed by the BER pathway, at least in bacterial and yeast cells.

Structural Insights into Phosphorylation-Mediated Polymerase Function Loss for DNA Polymerase ß Bound to Gapped DNA.

DNA polymerase ß is a member of the X-family of DNA polymerases, playing a critical role in the base excision repair (BER) pathway in mammalian cells by implementing the nucleotide gap-filling step. In vitro phosphorylation of DNA polymerase ß with PKC on S44 causes loss in the enzyme's DNA polymerase activity but not single-strand DNA binding. Although these studies have shown that single-stranded DNA binding is not affected by phosphorylation, the structural basis behind the mechanism underlying phosphorylation-induced activity loss remains poorly understood. Previous modeling studies suggested phosphorylation of S44 was sufficient to induce structural changes that impact the enzyme's polymerase function. However, the S44 phosphorylated-enzyme/DNA complex has not been modeled so far. To address this knowledge gap, we conducted atomistic molecular dynamics simulations of pol ß complexed with gapped DNA. Our simulations, which used explicit solvent and lasted for microseconds, revealed that phosphorylation at the S44 site, in the presence of Mg ions, induced significant conformational changes in the enzyme. Specifically, these changes led to the transformation of the enzyme from a closed to an open structure. Additionally, our simulations identified phosphorylation-induced allosteric coupling between the inter-domain region, suggesting the existence of a putative allosteric site. Taken together, our results provide a mechanistic understanding of the conformational transition observed due to phosphorylation in DNA polymerase ß interactions with gapped DNA. Our simulations shed light on the mechanisms of phosphorylation-induced activity loss in DNA polymerase ß and reveal potential targets for the development of novel therapeutics aimed at mitigating the effects of this post-translational modification.

Targeting DNA polymerase ß elicits synthetic lethality with mismatch repair deficiency in acute lymphoblastic leukemia.

Mismatch repair (MMR) deficiency has been linked to thiopurine resistance and hypermutation in relapsed acute lymphoblastic leukemia (ALL). However, the repair mechanism of thiopurine-induced DNA damage in the absence of MMR remains unclear. Here, we provide evidence that DNA polymerase ß (POLB) of base excision repair (BER) pathway plays a critical role in the survival and thiopurine resistance of MMR-deficient ALL cells. In these aggressive resistant ALL cells, POLB depletion and its inhibitor oleanolic acid (OA) treatment result in synthetic lethality with MMR deficiency through increased cellular apurinic/apyrimidinic (AP) sites, DNA strand breaks and apoptosis. POLB depletion increases thiopurine sensitivities of resistant cells, and OA synergizes with thiopurine to kill these cells in ALL cell lines, patient-derived xenograft (PDX) cells and xenograft mouse models. Our findings suggest BER and POLB's roles in the process of repairing thiopurine-induced DNA damage in MMR-deficient ALL cells, and implicate their potentials as therapeutic targets against aggressive ALL progression.

Visualizing the coordination of apurinic/apyrimidinic endonuclease (APE1) and DNA polymerase ß during base excision repair.

Base excision repair (BER) is carried out by a series of proteins that function in a step-by-step process to identify, remove, and replace DNA damage. During BER, the DNA transitions through various intermediate states as it is processed by each DNA repair enzyme. Left unrepaired, these BER intermediates can transition into double-stranded DNA breaks and promote genome instability. Previous studies have proposed a short-lived complex consisting of the BER intermediate, the incoming enzyme, and the outgoing enzyme at each step of the BER pathway to protect the BER intermediate. The transfer of BER intermediates between enzymes, known as BER coordination or substrate channeling, remains poorly understood. Here, we utilize single-molecule total internal reflection fluorescence microscopy to investigate the mechanism of BER coordination between apurinic/apyrimidinic endonuclease 1 (APE1) and DNA polymerase ß (Pol ß). When preformed complexes of APE1 and the incised abasic site product (APE1 product and Pol ß substrate) were subsequently bound by Pol ß, the Pol ß enzyme dissociated shortly after binding in most of the observations. In the events where Pol ß binding was followed by APE1 dissociation during substrate channeling, Pol ß remained bound for a longer period of time to allow disassociation of APE1. Our results indicate that transfer of the BER intermediate from APE1 to Pol ß during BER is dependent on the dissociation kinetics of APE1 and the duration of the ternary complex on the incised abasic site.

Fluorescently labeled human apurinic/apyrimidinic endonuclease APE1 reveals effects of DNA polymerase ß on the APE1-DNA interaction.

The base excision repair (BER) pathway involves sequential action of DNA glycosylases and apurinic/apyrimidinic (AP) endonucleases to incise damaged DNA and prepare DNA termini for incorporation of a correct nucleotide by DNA polymerases. It has been suggested that the enzymatic steps in BER include recognition of a product-enzyme complex by the next enzyme in the pathway, resulting in the "passing-the-baton" model of transfer of DNA intermediates between enzymes. To verify this model, in this work, we aimed to create a suitable experimental system. We prepared APE1 site-specifically labeled with a fluorescent reporter that is sensitive to stages of APE1-DNA binding, of formation of the catalytic complex, and of subsequent dissociation of the enzyme-product complex. Interactions of the labeled APE1 with various model DNA substrates (containing an abasic site) of varied lengths revealed that the enzyme remains mostly in complex with the DNA product. By means of the fluorescently labeled APE1 in combination with a stopped-flow fluorescence assay, it was found that Polß stimulates both i) APE1 binding to an abasic-site-containing DNA duplex with the formation of a catalytically competent complex and ii) the dissociation of APE1 from its product. These findings confirm DNA-mediated coordination of APE1 and Polß activities and suggest that Polß is the key trigger of the DNA transfer between the enzymes participating in initial steps of BER.

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.

Trypanosoma cruzi DNA Polymerase ß Is Phosphorylated In Vivo and In Vitro by Protein Kinase C (PKC) and Casein Kinase 2 (CK2).

DNA polymerase ß plays a fundamental role in the life cycle of Trypanosoma cruzi since it participates in the kinetoplast DNA repair and replication. This enzyme can be found in two forms in cell extracts of T. cruzi epimastigotes form. The H form is a phosphorylated form of DNA polymerase ß, while the L form is not phosphorylated. The protein kinases which are able to in vivo phosphorylate DNA polymerase ß have not been identified yet. In this work, we purified the H form of this DNA polymerase and identified the phosphorylation sites. DNA polymerase ß is in vivo phosphorylated at several amino acid residues including Tyr35, Thr123, Thr137 and Ser286. Thr123 is phosphorylated by casein kinase 2 and Thr137 and Ser286 are phosphorylated by protein kinase C-like enzymes. Protein kinase C encoding genes were identified in T. cruzi, and those genes were cloned, expressed in bacteria and the recombinant protein was purified. It was found that T. cruzi possesses three different protein kinase C-like enzymes named TcPKC1, TcPKC2, and TcPKC3. Both TcPKC1 and TcPKC2 were able to in vitro phosphorylate recombinant DNA polymerase ß, and in addition, TcPKC1 gets auto phosphorylated. Those proteins contain several regulatory domains at the N-terminus, which are predicted to bind phosphoinositols, and TcPKC1 contains a lipocalin domain at the C-terminus that might be able to bind free fatty acids. Tyr35 is phosphorylated by an unidentified protein kinase and considering that the T. cruzi genome does not contain Tyr kinase encoding genes, it is probable that Tyr35 could be phosphorylated by a dual protein kinase. Wee1 is a eukaryotic dual protein kinase involved in cell cycle regulation. We identified a Wee1 homolog in T. cruzi and the recombinant kinase was assayed using DNA polymerase ß as a substrate. T. cruzi Wee1 was able to in vitro phosphorylate recombinant DNA polymerase ß, although we were not able to demonstrate specific phosphorylation on Tyr35. Those results indicate that there exists a cell signaling pathway involving PKC-like kinases in T. cruzi.

Polß modulates the expression of type I interferon via STING pathway.

DNA Polymerase ß (Polß) is a key enzyme in base excision repair (BER), which is very important in maintaining the stability and integrity of the genome. Mutant Polß is closely associated with carcinogenesis. However, Polß is highly expressed in most cancers, but the underlying mechanism is not well understood. Here, we found that breast cancer cells MCF-7 with Polß knockdown exhibited high levels of type I interferon and were easily eliminated by natural killer (NK) cells.Similarly, Polß-mutant (R137Q) mice exhibited chronic inflammation symptoms in multiple organs and upregulated type I interferon levels. Further results showed that Polß deficiency caused more DNA damage accumulation in cells and triggered the leakage of damaged DNA into the cytoplasm, which activated the STING/IRF3 pathway, promoted phosphorylated IRF3 translocating into the nucleus and enhanced the expression of type I interferon and proinflammatory cytokines. In addition, this effect could be eliminated by Polß overexpression, STING inhibitor or STING knockdown. Taken together, our findings provide mechanistic insight into the role of Polß in cancers by linking DNA repair and the inflammatory STING pathway.