DNA Repair Protein XRCC1 Stimulates Activity of DNA Polymerase λ under Conditions of Microphase Separation.

Non-membrane compartments or biomolecular condensates play an important role in the regulation of cellular processes including DNA repair. Here, an ability of XRCC1, a scaffold protein involved in DNA base excision repair (BER) and single-strand break repair, to form protein-rich microphases in the presence of DNA duplexes was discovered. We also showed that the gap-filling activity of BER-related DNA polymerase λ (Pol λ) is significantly increased by the presence of XRCC1. The stimulation of the Pol λ activity was observed only at micromolar XRCC1 concentrations, which were well above the nanomolar dissociation constant determined for the XRCC1-Pol λ complex and pointed to the presence of an auxiliary stimulatory factor in addition to protein-protein interactions. Indeed, according to dynamic light scattering measurements, the stimulation of the Pol λ activity by XRCC1 was coupled with microphase separation in a protein-DNA mixture. Fluorescence microscopy revealed colocalization of Pol λ, XRCC1, and gapped DNA within the microphases. Thus, stimulation of Pol λ activity is caused both by its interaction with XRCC1 and by specific conditions of microphase separation; this phenomenon is shown for the first time.

Correct Nucleotide Selection Is Confined at the Binding Site of Polymerase Enzymes.

DNA polymerases (Pols) add incoming nucleotides (deoxyribonucleoside triphosphate (dNTPs)) to growing DNA strands, a crucial step for DNA synthesis. The insertion of correct (vs incorrect) nucleotides relates to Pols' fidelity, which defines Pols' ability to faithfully replicate DNA strands in a template-dependent manner. We and others have demonstrated that reactant alignment and correct base pairing at the Pols catalytic site are crucial structural features to fidelity. Here, we first used equilibrium molecular simulations to demonstrate that the local dynamics at the protein-DNA interface in the proximity of the catalytic site is different when correct vs incorrect dNTPs are bound to polymerase ß (Pol ß). Formation and dynamic stability of specific interatomic interactions around the incoming nucleotide influence the overall binding site architecture. This explains why certain Pols' mutants can affect the local catalytic environment and influence the selection of correct vs incorrect nucleotides. In particular, this is here demonstrated by analyzing the interaction network formed by the residue R283, whose mutant R283A has an experimentally measured lower capacity of differentiating correct (G:dCTP) vs incorrect (G:dATP) base pairing in Pol ß. We also used alchemical free-energy calculations to quantify the G:dCTP →G:dATP transformation in Pol ß wild-type and mutant R283A. These results correlate well with the experimental trend, thus corroborating our mechanistic insights. Sequence and structural comparisons with other Pols from the same family suggest that these findings may also be valid in similar enzymes.

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.

Probing the Conformational Restraints of DNA Damage Recognition with ß-L-Nucleotides.

The DNA building blocks 2'-deoxynucleotides are enantiomeric, with their natural ß-D-configuration dictated by the sugar moiety. Their synthetic ß-L-enantiomers (ßLdNs) can be used to obtain L-DNA, which, when fully substituted, is resistant to nucleases and is finding use in many biosensing and nanotechnology applications. However, much less is known about the enzymatic recognition and processing of individual ßLdNs embedded in D-DNA. Here, we address the template properties of ßLdNs for several DNA polymerases and the ability of base excision repair enzymes to remove these modifications from DNA. The Klenow fragment was fully blocked by ßLdNs, whereas DNA polymerase κ bypassed them in an error-free manner. Phage RB69 DNA polymerase and DNA polymerase ß treated ßLdNs as non-instructive but the latter enzyme shifted towards error-free incorporation on a gapped DNA substrate. DNA glycosylases and AP endonucleases did not process ßLdNs. DNA glycosylases sensitive to the base opposite their cognate lesions also did not recognize ßLdNs as a correct pairing partner. Nevertheless, when placed in a reporter plasmid, pyrimidine ßLdNs were resistant to repair in human cells, whereas purine ßLdNs appear to be partly repaired. Overall, ßLdNs are unique modifications that are mostly non-instructive but have dual non-instructive/instructive properties in special cases.

Impact of DNA ligase 1 and IIIα interactions with APE1 and polß on the efficiency of base excision repair pathway at the downstream steps.

Base excision repair (BER) requires a tight coordination between the repair enzymes through protein-protein interactions and involves gap filling by DNA polymerase (pol) ß and subsequent nick sealing by DNA ligase (LIG) 1 or LIGIIIα at the downstream steps. Apurinic/apyrimidinic-endonuclease 1 (APE1), by its exonuclease activity, proofreads 3' mismatches incorporated by polß during BER. We previously reported that the interruptions in the functional interplay between polß and the BER ligases result in faulty repair events. Yet, how the protein interactions of LIG1 and LIGIIIα could affect the repair pathway coordination during nick sealing at the final steps remains unknown. Here, we demonstrate that LIGIIIα interacts more tightly with polß and APE1 than LIG1, and the N-terminal noncatalytic region of LIG1 as well as the catalytic core and BRCT domain of LIGIIIα mediate interactions with both proteins. Our results demonstrated less efficient nick sealing of polß nucleotide insertion products in the absence of LIGIIIα zinc-finger domain and LIG1 N-terminal region. Furthermore, we showed a coordination between APE1 and LIG1/LIGIIIα during the removal of 3' mismatches from the nick repair intermediate on which both BER ligases can seal noncanonical ends or gap repair intermediate leading to products of single deletion mutagenesis. Overall results demonstrate the importance of functional coordination from gap filling by polß coupled to nick sealing by LIG1/LIGIIIα in the presence of proofreading by APE1, which is mainly governed by protein-protein interactions and protein-DNA intermediate communications, to maintain repair efficiency at the downstream steps of the BER pathway.

Modifying the Basicity of the dNTP Leaving Group Modulates Precatalytic Conformational Changes of DNA Polymerase ß.

The catalytic function of DNA polymerase ß (pol ß) fulfills the gap-filling requirement of the base excision DNA repair pathway by incorporating a single nucleotide into a gapped DNA substrate resulting from the removal of damaged DNA bases. Most importantly, pol ß can select the correct nucleotide from a pool of similarly structured nucleotides to incorporate into DNA in order to prevent the accumulation of mutations in the genome. Pol ß is likely to employ various mechanisms for substrate selection. Here, we use dCTP analogues that have been modified at the ß,γ-bridging group of the triphosphate moiety to monitor the effect of leaving group basicity of the incoming nucleotide on precatalytic conformational changes, which are important for catalysis and selectivity. It has been previously shown that there is a linear free energy relationship between leaving group pKa and the chemical transition state. Our results indicate that there is a similar relationship with the rate of a precatalytic conformational change, specifically, the closing of the fingers subdomain of pol ß. In addition, by utilizing analogue ß,γ-CHX stereoisomers, we identified that the orientation of the ß,γ-bridging group relative to R183 is important for the rate of fingers closing, which directly influences chemistry.

Oxidatively-induced DNA base damage and base excision repair abnormalities in siblings of individuals with bipolar disorder DNA damage and repair in bipolar disorder.

Previous evidence suggests elevated levels of oxidatively-induced DNA damage, particularly 8-hydroxy-2'-deoxyguanosine (8-OH-dG), and abnormalities in the repair of 8-OH-dG by the base excision repair (BER) in bipolar disorder (BD). However, the genetic disposition of these abnormalities remains unknown. In this study, we aimed to investigate the levels of oxidatively-induced DNA damage and BER mechanisms in individuals with BD and their siblings, as compared to healthy controls (HCs). 46 individuals with BD, 41 siblings of individuals with BD, and 51 HCs were included in the study. Liquid chromatography-tandem mass spectrometry was employed to evaluate the levels of 8-OH-dG in urine, which were then normalized based on urine creatinine levels. The real-time-polymerase chain reaction was used to measure the expression levels of 8-oxoguanine DNA glycosylase 1 (OGG1), apurinic/apyrimidinic endonuclease 1 (APE1), poly ADP-ribose polymerase 1 (PARP1), and DNA polymerase beta (POLß). The levels of 8-OH-dG were found to be elevated in both individuals with BD and their siblings when compared to the HCs. The OGG1 and APE1 expressions were downregulated, while POLß expressions were upregulated in both the patient and sibling groups compared to the HCs. Age, smoking status, and the number of depressive episodes had an impact on APE1 expression levels in the patient group while body mass index, smoking status, and past psychiatric history had an impact on 8-OH-dG levels in siblings. Both individuals with BD and unaffected siblings presented similar abnormalities regarding oxidatively-induced DNA damage and BER, suggesting a link between abnormalities in DNA damage/BER mechanisms and familial susceptibility to BD. Our findings suggest that targeting the oxidatively-induced DNA damage and BER pathway could offer promising therapeutic strategies for reducing the risk of age-related diseases and comorbidities in individuals with a genetic predisposition to BD.

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.

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.

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.

Biochemical and structural characterization of Fapy•dG replication by Human DNA polymerase ß.

N6-(2-deoxy-α,ß-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido-pyrimidine (Fapy•dG) is formed from a common intermediate and in comparable amounts to the well-studied mutagenic DNA lesion 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodGuo). Fapy•dG preferentially gives rise to G → T transversions and G → A transitions. However, the molecular basis by which Fapy•dG is processed by DNA polymerases during this mutagenic process remains poorly understood. To address this we investigated how DNA polymerase ß (Pol ß), a model mammalian polymerase, bypasses a templating Fapy•dG, inserts Fapy•dGTP, and extends from Fapy•dG at the primer terminus. When Fapy•dG is present in the template, Pol ß incorporates TMP less efficiently than either dCMP or dAMP. Kinetic analysis revealed that Fapy•dGTP is a poor substrate but is incorporated ∼3-times more efficiently opposite dA than dC. Extension from Fapy•dG at the 3'-terminus of a nascent primer is inefficient due to the primer terminus being poorly positioned for catalysis. Together these data indicate that mutagenic bypass of Fapy•dG is likely to be the source of the mutagenic effects of the lesion and not Fapy•dGTP. These experiments increase our understanding of the promutagenic effects of Fapy•dG.

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.

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.

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.

Human DNA ligases I and IIIα as determinants of accuracy and efficiency of base excision DNA repair.

Mammalian Base Excision Repair (BER) DNA ligases I and IIIα (LigI, LigIIIα) are major determinants of DNA repair fidelity, alongside with DNA polymerases. Here we compared activities of human LigI and LigIIIα on specific and nonspecific substrates representing intermediates of distinct BER sub-pathways. The enzymes differently discriminate mismatches in the nicked DNA, depending on their identity and position, but are both more selective against the 3'-end non-complementarity. LigIIIα is less active than LigI in premature ligation of one-nucleotide gapped DNA and more efficiently discriminates misinsertion products of DNA polymerase ß-catalyzed gap filling, that reinforces a leading role of LigIIIα in the accuracy of short-patch BER. LigI and LigIIIα reseal the intermediate of long-patch BER containing an incised synthetic AP site (F) with different efficiencies, depending on the DNA sequence context, 3'-end mismatch presence and coupling of the ligation reaction with DNA repair synthesis. Processing of this intermediate in the absence of flap endonuclease 1 generates non-canonical DNAs with bulged F site, which are very inefficiently repaired by AP endonuclease 1 and represent potential mutagenic repair products. The extent of conversion of the 5'-adenylated intermediates of specific and nonspecific substrates is revealed to depend on the DNA sequence context; a higher sensitivity of LigI to the sequence is in line with the enzyme structural feature of DNA binding. LigIIIα exceeds LigI in generation of potential abortive ligation products, justifying importance of XRCC1-mediated coordination of LigIIIα and aprataxin activities for the efficient DNA repair.

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.