Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs.

We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.

A Small-Molecule Inhibitor of Human DNA Polymerase η Potentiates the Effects of Cisplatin in Tumor Cells.

Translesion DNA synthesis (TLS) performed by human DNA polymerase eta (hpol η) allows tolerance of damage from cis-diamminedichloroplatinum(II) (CDDP or cisplatin). We have developed hpol η inhibitors derived from N-aryl-substituted indole barbituric acid (IBA), indole thiobarbituric acid (ITBA), and indole quinuclidine scaffolds and identified 5-((5-chloro-1-(naphthalen-2-ylmethyl)-1H-indol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (PNR-7-02), an ITBA derivative that inhibited hpol η activity with an IC50 value of 8 µM and exhibited 5-10-fold specificity for hpol η over replicative pols. We conclude from kinetic analyses, chemical footprinting assays, and molecular docking that PNR-7-02 binds to a site on the little finger domain and interferes with the proper orientation of template DNA to inhibit hpol η. A synergistic increase in CDDP toxicity was observed in hpol η-proficient cells co-treated with PNR-7-02 (combination index values = 0.4-0.6). Increased γH2AX formation accompanied treatment of hpol η-proficient cells with CDDP and PNR-7-02. Importantly, PNR-7-02 did not impact the effect of CDDP on cell viability or γH2AX in hpol η-deficient cells. In summary, we observed hpol η-dependent effects on DNA damage/replication stress and sensitivity to CDDP in cells treated with PNR-7-02. The ability to employ a small-molecule inhibitor of hpol η to improve the cytotoxic effect of CDDP may aid in the development of more effective chemotherapeutic strategies.

Evidence That G-quadruplex DNA Accumulates in the Cytoplasm and Participates in Stress Granule Assembly in Response to Oxidative Stress.

Cells engage numerous signaling pathways in response to oxidative stress that together repair macromolecular damage or direct the cell toward apoptosis. As a result of DNA damage, mitochondrial DNA or nuclear DNA has been shown to enter the cytoplasm where it binds to "DNA sensors," which in turn initiate signaling cascades. Here we report data that support a novel signaling pathway in response to oxidative stress mediated by specific guanine-rich sequences that can fold into G-quadruplex DNA (G4DNA). In response to oxidative stress, we demonstrate that sequences capable of forming G4DNA appear at increasing levels in the cytoplasm and participate in assembly of stress granules. Identified proteins that bind to endogenous G4DNA in the cytoplasm are known to modulate mRNA translation and participate in stress granule formation. Consistent with these findings, stress granule formation is known to regulate mRNA translation during oxidative stress. We propose a signaling pathway whereby cells can rapidly respond to DNA damage caused by oxidative stress. Guanine-rich sequences that are excised from damaged genomic DNA are proposed to enter the cytoplasm where they can regulate translation through stress granule formation. This newly proposed role for G4DNA provides an additional molecular explanation for why such sequences are prevalent in the human genome.

Human Translesion Polymerase κ Exhibits Enhanced Activity and Reduced Fidelity Two Nucleotides from G-Quadruplex DNA.

We have investigated the in vitro properties of human Y-family polymerase κ (hpol κ) on G-quadruplex DNA (G4 DNA). Similar to hpol η, another Y-family member implicated in replication of G4 motifs, hpol κ bound G4 DNA with a 5.7-fold preference over control, non-G4 DNA. Results from pol extension assays are consistent with the notion that G-quadruplexes present a stronger barrier to DNA synthesis by hpol κ than they do to that by hpol η. However, kinetic analysis revealed that hpol κ activity increases considerably when the enzyme is 2-3 nucleotides from the G4 motif, a trend that was reported previously for hpol η, though the increase was less pronounced. The increase in hpol κ activity on G4 DNA was readily observed in the presence of either potassium or sodium but much less so when lithium was used in the buffer. The increased activity 2-3 nucleotides from the G4 motif was accompanied by a decrease in the fidelity of hpol κ when the counterion was either potassium or sodium but not in the presence of lithium. The activity of hpol κ decreased progressively as the primer was moved closer than 2 nucleotides from the G4 motif when either potassium or sodium was used to stabilize the G-quadruplex. Interestingly, the decrease in catalytic activity at the site of the quadruplex observed in potassium-containing buffer was accompanied by an increase in fidelity on G4 substrates versus control non-G4 substrates. This trend of increased fidelity in copying a tetrad-associated guanine was observed previously for hpol η, but not for the B-family member hpol ε, which exhibited a large decrease in both efficiency and fidelity in the attempt to copy the first guanine in the G4 motif. In summary, hpol κ activity was enhanced relative to those of other Y-family members when the enzyme is 2-3 nucleotides from the G4 motif, but hpol κ appears to be less competent than hpol η at copying tetrad-associated guanines.

Kynurenine Signaling Increases DNA Polymerase Kappa Expression and Promotes Genomic Instability in Glioblastoma Cells.

Overexpression of the translesion synthesis polymerase hpol κ in glioblastomas has been linked to poor patient prognosis; however, the mechanism promoting higher expression in these tumors remains unknown. We determined that activation of the aryl hydrocarbon receptor (AhR) pathway in glioblastoma cells leads to increased hpol κ mRNA and protein levels. We blocked nuclear translocation and DNA binding by AhR in glioblastoma cells using a small-molecule and observed decreased hpol κ expression. Pharmacological inhibition of tryptophan-2,3-dioxygenase (TDO), the enzyme largely responsible for activating AhR in glioblastoma, led to a decrease in the endogenous AhR agonist kynurenine and a corresponding decrease in hpol κ protein levels. Importantly, we discovered that inhibiting TDO activity, AhR signaling, or suppressing hpol κ expression with RNA interference led to decreased chromosomal damage in glioblastoma cells. Epistasis assays further supported the idea that TDO activity, activation of AhR signaling, and the resulting overexpression of hpol κ function primarily in the same pathway to increase endogenous DNA damage. These findings indicate that upregulation of hpol κ through glioblastoma-specific TDO activity and activation of AhR signaling likely contributes to the high levels of replication stress and genomic instability observed in these tumors.

The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa.

Human DNA polymerase kappa (hpol κ) is the only Y-family member to preferentially insert dAMP opposite 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) during translesion DNA synthesis. We have studied the mechanism of action by which hpol κ activity is modulated by the Werner syndrome protein (WRN), a RecQ helicase known to influence repair of 8-oxo-dG. Here we show that WRN stimulates the 8-oxo-dG bypass activity of hpol κ in vitro by enhancing the correct base insertion opposite the lesion, as well as extension from dC:8-oxo-dG base pairs. Steady-state kinetic analysis reveals that WRN improves hpol κ-catalyzed dCMP insertion opposite 8-oxo-dG ∼10-fold and extension from dC:8-oxo-dG by 2.4-fold. Stimulation is primarily due to an increase in the rate constant for polymerization (kpol), as assessed by pre-steady-state kinetics, and it requires the RecQ C-terminal (RQC) domain. In support of the functional data, recombinant WRN and hpol κ were found to physically interact through the exo and RQC domains of WRN, and co-localization of WRN and hpol κ was observed in human cells treated with hydrogen peroxide. Thus, WRN limits the error-prone bypass of 8-oxo-dG by hpol κ, which could influence the sensitivity to oxidative damage that has previously been observed for Werner's syndrome cells.

Human Rev1 polymerase disrupts G-quadruplex DNA.

The Y-family DNA polymerase Rev1 is required for successful replication of G-quadruplex DNA (G4 DNA) in higher eukaryotes. Here we show that human Rev1 (hRev1) disrupts G4 DNA structures and prevents refolding in vitro. Nucleotidyl transfer by hRev1 is not necessary for mechanical unfolding to occur. hRev1 binds G4 DNA substrates with Kd,DNA values that are 4-15-fold lower than those of non-G4 DNA substrates. The pre-steady-state rate constant of deoxycytidine monophosphate (dCMP) insertion opposite the first tetrad-guanine by hRev1 is ∼56% as fast as that observed for non-G4 DNA substrates. Thus, hRev1 can promote fork progression by either dislodging tetrad guanines to unfold the G4 DNA, which could assist in extension by other DNA polymerases, or hRev1 can prevent refolding of G4 DNA structures. The hRev1 mechanism of action against G-quadruplexes helps explain why replication progress is impeded at G4 DNA sites in Rev1-deficient cells and illustrates another unique feature of this enzyme with important implications for genome maintenance.

Evidence for the kinetic partitioning of polymerase activity on G-quadruplex DNA.

We have investigated the action of the human DNA polymerase ε (hpol ε) and η (hpol η) catalytic cores on G-quadruplex (G4) DNA substrates derived from the promoter of the c-MYC proto-oncogene. The translesion enzyme hpol η exhibits a 6.2-fold preference for binding to G4 DNA over non-G4 DNA, while hpol ε binds both G4 and non-G4 substrates with nearly equal affinity. Kinetic analysis of single-nucleotide insertion by hpol η reveals that it is able to maintain >25% activity on G4 substrates compared to non-G4 DNA substrates, even when the primer template junction is positioned directly adjacent to G22 (the first tetrad-associated guanine in the c-MYC G4 motif). Surprisingly, hpol η fidelity increases ~15-fold when copying G22. By way of comparison, hpol ε retains ~4% activity and has a 33-fold decrease in fidelity when copying G22. The fidelity of hpol η is ~100-fold greater than that of hpol ε when comparing the misinsertion frequencies of the two enzymes opposite a tetrad-associated guanine. The kinetic differences observed for the B- and Y-family pols on G4 DNA support a model in which a simple kinetic switch between replicative and TLS pols could help govern fork progress during G4 DNA replication.

Replication, repair, and translesion polymerase bypass of N6-oxopropenyl-2'-deoxyadenosine.

The oxidative stress products malondialdehyde and base propenal react with DNA bases forming the adduction products 3-(2'-deoxy-ß-D-erythro-pentofuranosyl)pyrimido[1,2-a]purin-10(3H)-one (M1dG) and N(6)-(oxypropenyl)-2'-deoxyadenosine (OPdA). M1dG is mutagenic in vivo and miscodes in vitro, but little work has been done on OPdA. To improve our understanding of the effect of OPdA on polymerase activity and mutagenicity, we evaluated the ability of the translesion DNA polymerases hPols η, κ, and ι to bypass OPdA in vitro. hPols η and κ inserted dNTPs opposite the lesion and extended the OPdA-modified primer to the terminus. hPol ι inserted dNTPs opposite OPdA but failed to fully extend the primer. Steady-state kinetic analysis indicated that these polymerases preferentially insert dTTP opposite OPdA, although less efficiently than opposite dA. Minimal incorrect base insertion was observed for all polymerases, and dCTP was the primary mis-insertion event. Examining replicative and repair polymerases revealed little effect of OPdA on the Sulfolobus solfataricus polymerase Dpo1 or the Klenow fragment of Escherichia coli DNA polymerase I. Bacteriophage T7 DNA polymerase displayed a reduced level of OPdA bypass compared to unmodified DNA, and OPdA nearly completely blocked the activity of base excision repair polymerase hPol ß. This work demonstrates that bypass of OPdA is generally error-free, modestly decreases the catalytic activity of most polymerases, and blocks hPol ß polymerase activity. Although mis-insertion opposite OPdA is relatively weak, the efficiency of bypass may introduce A → G transitions observed in vivo.

Enhancement of human DNA polymerase η activity and fidelity is dependent upon a bipartite interaction with the Werner syndrome protein.

We have investigated the interaction between human DNA polymerase η (hpol η) and the Werner syndrome protein (WRN). Functional assays revealed that the WRN exonuclease and RecQ C-terminal (RQC) domains are necessary for full stimulation of hpol η-catalyzed formation of correct base pairs. We find that WRN does not stimulate hpol η-catalyzed formation of mispairs. Moreover, the exonuclease activity of WRN prevents stable mispair formation by hpol η. These results are consistent with a proofreading activity for WRN during single-nucleotide additions. ATP hydrolysis by WRN appears to attenuate stimulation of hpol η. Pre-steady-state kinetic results show that k(pol) is increased 4-fold by WRN. Finally, pulldown assays reveal a bipartite physical interaction between hpol η and WRN that is mediated by the exonuclease and RQC domains. Taken together, these results are consistent with alteration of the rate-limiting step in polymerase catalysis by direct protein-protein interactions between WRN and hpol η. In summary, WRN improves the efficiency and fidelity of hpol η to promote more effective replication of DNA.

Translesion synthesis across 1,N6-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N6-γ-HMHP-dA) adducts by human and archebacterial DNA polymerases.

The 1,N(6)-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N(6)-γ-HMHP-dA) adducts are formed upon bifunctional alkylation of adenine nucleobases in DNA by 1,2,3,4-diepoxybutane, the putative ultimate carcinogenic metabolite of 1,3-butadiene. The presence of a substituted 1,N(6)-propano group on 1,N(6)-γ-HMHP-dA is expected to block the Watson-Crick base pairing of the adducted adenine with thymine, potentially contributing to mutagenesis. In this study, the enzymology of replication past site-specific 1,N(6)-γ-HMHP-dA lesions in the presence of human DNA polymerases (hpols) ß, η, κ, and ι and archebacterial polymerase Dpo4 was investigated. Run-on gel analysis with all four dNTPs revealed that hpol η, κ, and Dpo4 were able to copy the modified template. In contrast, hpol ι inserted a single base opposite 1,N(6)-γ-HMHP-dA but was unable to extend beyond the damaged site, and a complete replication block was observed with hpol ß. Single nucleotide incorporation experiments indicated that although hpol η, κ, and Dpo4 incorporated the correct nucleotide (dTMP) opposite the lesion, dGMP and dAMP were inserted with a comparable frequency. HPLC-ESI-MS/MS analysis of primer extension products confirmed the ability of bypass polymerases to insert dTMP, dAMP, or dGMP opposite 1,N(6)-γ-HMHP-dA and detected large amounts of -1 and -2 deletion products. Taken together, these results indicate that hpol η and κ enzymes bypass 1,N(6)-γ-HMHP-dA lesions in an error-prone fashion, potentially contributing to A→T and A→C transversions and frameshift mutations observed in cells following treatment with 1,2,3,4-diepoxybutane.

Leukotriene biosynthesis inhibitor MK886 impedes DNA polymerase activity.

Specialized DNA polymerases participate in replication stress responses and in DNA repair pathways that function as barriers against cellular senescence and genomic instability. These events can be co-opted by tumor cells as a mechanism to survive chemotherapeutic and ionizing radiation treatments and as such, represent potential targets for adjuvant therapies. Previously, a high-throughput screen of ∼16,000 compounds identified several first generation proof-of-principle inhibitors of human DNA polymerase kappa (hpol κ). The indole-derived inhibitor of 5-lipoxygenase activating protein (FLAP), MK886, was one of the most potent inhibitors of hpol κ discovered in that screen. However, the specificity and mechanism of inhibition remained largely undefined. In the current study, the specificity of MK886 against human Y-family DNA polymerases and a model B-family DNA polymerase was investigated. MK886 was found to inhibit the activity of all DNA polymerases tested with similar IC(50) values, the exception being a 6- to 8-fold increase in the potency of inhibition against human DNA polymerase iota (hpol ι), a highly error-prone enzyme that uses Hoogsteen base-pairing modes during catalysis. The specificity against hpol ι was partially abrogated by inclusion of the recently annotated 25 a.a. N-terminal extension. On the basis of Michaelis-Menten kinetic analyses and DNA binding assays, the mechanism of inhibition by MK886 appears to be mixed. In silico docking studies were used to produce a series of models for MK886 binding to Y-family members. The docking results indicate that two binding pockets are conserved between Y-family polymerases, while a third pocket near the thumb domain appears to be unique to hpol ι. Overall, these results provide insight into the general mechanism of DNA polymerase inhibition by MK886.

Inhibition of tryptophan 2,3-dioxygenase impairs DNA damage tolerance and repair in glioma cells.

Expression of tryptophan 2,3-dioxygenase (TDO) is a determinant of malignancy in gliomas through kynurenine (KYN) signaling. We report that inhibition of TDO activity attenuated recovery from replication stress and increased the genotoxic effects of bis-chloroethylnitrosourea (BCNU). Activation of the Chk1 arm of the replication stress response (RSR) was reduced when TDO activity was blocked prior to BCNU treatment, whereas phosphorylation of serine 33 (pS33) on replication protein A (RPA) was enhanced-indicative of increased fork collapse. Analysis of quantitative proteomic results revealed that TDO inhibition reduced nuclear 53BP1 and sirtuin levels. We confirmed that cells lacking TDO activity exhibited elevated gamma-H2AX signal and defective recruitment of 53BP1 to chromatin following BCNU treatment, which corresponded with delayed repair of DNA breaks. Addition of exogenous KYN increased the rate of break repair. TDO inhibition diminished SIRT7 deacetylase recruitment to chromatin, which increased histone H3K18 acetylation-a key mark involved in preventing 53BP1 recruitment to sites of DNA damage. TDO inhibition also sensitized cells to ionizing radiation (IR)-induced damage, but this effect did not involve altered 53BP1 recruitment. These experiments support a model where TDO-mediated KYN signaling helps fuel a robust response to replication stress and DNA damage.

A Functional Precision Medicine Pipeline Combines Comparative Transcriptomics and Tumor Organoid Modeling to Identify Bespoke Treatment Strategies for Glioblastoma.

Li Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome caused by germline mutations in TP53. TP53 is the most common mutated gene in human cancer, occurring in 30-50% of glioblastomas (GBM). Here, we highlight a precision medicine platform to identify potential targets for a GBM patient with LFS. We used a comparative transcriptomics approach to identify genes that are uniquely overexpressed in the LFS GBM patient relative to a cancer compendium of 12,747 tumor RNA sequencing data sets, including 200 GBMs. STAT1 and STAT2 were identified as being significantly overexpressed in the LFS patient, indicating ruxolitinib, a Janus kinase 1 and 2 inhibitors, as a potential therapy. The LFS patient had the highest level of STAT1 and STAT2 expression in an institutional high-grade glioma cohort of 45 patients, further supporting the cancer compendium results. To empirically validate the comparative transcriptomics pipeline, we used a combination of adherent and organoid cell culture techniques, including ex vivo patient-derived organoids (PDOs) from four patient-derived cell lines, including the LFS patient. STAT1 and STAT2 expression levels in the four patient-derived cells correlated with levels identified in the respective parent tumors. In both adherent and organoid cultures, cells from the LFS patient were among the most sensitive to ruxolitinib compared to patient-derived cells with lower STAT1 and STAT2 expression levels. A spheroid-based drug screening assay (3D-PREDICT) was performed and used to identify further therapeutic targets. Two targeted therapies were selected for the patient of interest and resulted in radiographic disease stability. This manuscript supports the use of comparative transcriptomics to identify personalized therapeutic targets in a functional precision medicine platform for malignant brain tumors.

In vitro bypass of the major malondialdehyde- and base propenal-derived DNA adduct by human Y-family DNA polymerases κ, ι, and Rev1.

3-(2'-Deoxy-ß-d-erythro-pentofuranosyl)pyrimido-[1,2-a]purin-10(3H)-one (M(1)dG) is the major adduct derived from the reaction of DNA with the lipid peroxidation product malondialdehyde and the DNA peroxidation product base propenal. M(1)dG is mutagenic in Escherichia coli and mammalian cells, inducing base-pair substitutions (M(1)dG → A and M(1)dG → T) and frameshift mutations. Y-family polymerases may contribute to the mutations induced by M(1)dG in vivo. Previous reports described the bypass of M(1)dG by DNA polymerases η and Dpo4. The present experiments were conducted to evaluate bypass of M(1)dG by the human Y-family DNA polymerases κ, ι, and Rev1. M(1)dG was incorporated into template-primers containing either dC or dT residues 5' to the adduct, and the template-primers were subjected to in vitro replication by the individual DNA polymerases. Steady-state kinetic analysis of single nucleotide incorporation indicates that dCMP is most frequently inserted by hPol κ opposite the adduct in both sequence contexts, followed by dTMP and dGMP. dCMP and dTMP were most frequently inserted by hPol ι, and only dCMP was inserted by Rev1. hPol κ extended template-primers in the order M(1)dG:dC > M(1)dG:dG > M(1)dG:dT ∼ M(1)dG:dA, but neither hPol ι nor Rev1 extended M(1)dG-containing template-primers. Liquid chromatography-mass spectrometry analysis of the products of hPol κ-catalyzed extension verified this preference in the 3'-GXC-5' template sequence but revealed the generation of a series of complex products in which dAMP is incorporated opposite M(1)dG in the 3'-GXT-5' template sequence. The results indicate that DNA hPol κ or the combined action of hPol ι or Rev1 and hPol κ bypass M(1)dG residues in DNA and generate products that are consistent with some of the mutations induced by M(1)dG in mammalian cells.

Cockayne syndrome group B protein is engaged in processing of DNA adducts of lipid peroxidation product trans-4-hydroxy-2-nonenal.

Cockayne syndrome complementation group B (CSB) protein is engaged in transcription-coupled repair (TCR) of UV induced DNA damage and its deficiency leads to progressive multisystem degeneration and premature aging. Here, we show that human CSB-deficient cells are hypersensitive to physiological concentrations (1-10 microM) of a lipid peroxidation product, trans-4-hydroxy-2-nonenal (HNE), and in response to HNE they develop a higher level of sister chromatid exchanges (SCEs) in comparison to the wild-type cells. HNE-DNA adducts block in vitro transcription by T7 RNA polymerase, as well as by HeLa cell-free extracts. Treatment of wild-type cells with 1-20 microM HNE causes dephosphorylation of the CSB protein, which stimulates its ATPase activity necessary for TCR. However, high HNE concentrations (100-200 microM) inhibit in vitro CSB ATPase activity as well as the transcription machinery in HeLa cell-free extracts. Cell lines expressing CSB protein mutated in different ATPase domains exhibit different sensitivities to HNE. The motif II mutant, which binds ATP, but is defective in ATP hydrolysis was as sensitive to HNE as CSB-null cells. In contrast, motif V mutant cells were as sensitive to HNE as were the cells bearing wild-type protein, while motif VI mutant cells showed intermediate sensitivity to HNE. These mutants exhibit decreased ATP binding, but retain residual ATPase activity. Homology modeling suggested that amino acids mutated in motifs II and VI are localized closer to the ATP binding site than amino acids mutated in ATPase motif V. These results suggest that HNE-DNA adducts are extremely toxic endogenous DNA lesion, and that their processing involves CSB. When these lesions are not removed from the transcribed DNA strand due to CSB gene mutation or CSB protein inactivation by high, pathological HNE concentrations, they may contribute to accelerated aging.

Inhibition of Human DNA Polymerases Eta and Kappa by Indole-Derived Molecules Occurs through Distinct Mechanisms.

Overexpression of human DNA polymerase kappa (hpol κ) in glioblastoma is associated with shorter survival time and resistance to the alkylating agent temozolomide (TMZ), making it an attractive target for the development of small-molecule inhibitors. We previously reported on the development and characterization of indole barbituric acid-derived (IBA) inhibitors of translesion DNA synthesis polymerases (TLS pols). We have now identified a potent and selective inhibitor of hpol κ based on the indole-aminoguanidine (IAG) chemical scaffold. The most promising IAG analogue, IAG-10, exhibited greater inhibitory action against hpol κ than any other human Y-family member, as well as pols from the A-, B-, and X-families. Inhibition of hpol κ by IAG analogues appears to proceed through a mechanism that is distinct from inhibition of hpol η based on changes in DNA binding affinity and nucleotide insertion kinetics. By way of comparison, both IAG and IBA analogues inhibited binary complex formation by hpol κ and ternary complex formation by hpol η. Decreasing the concentration of enzyme and DNA in the reaction mixture lowered the IC50 value of IAG-10 to submicromolar values, consistent with inhibition of binary complex formation for hpol κ. Chemical footprinting experiments revealed that IAG-10 binds to a cleft between the finger, little finger, and N-clasp domains on hpol κ and that this likely disrupts the interaction between the N-clasp and the TLS pol core. In cell culture, IAG-10 potentiated the antiproliferative activity and DNA damaging effects of TMZ in hpol κ-proficient cells but not in hpol κ-deficient cells, indicative of a target-dependent effect. Mutagenic replication across alkylation damage increased in hpol κ-proficient cells treated with IAG-10, while no change in mutation frequency was observed for hpol κ-deficient cells. In summary, we developed a potent and selective small-molecule inhibitor of hpol κ that takes advantage of structural features unique to this TLS enzyme to potentiate TMZ, a standard-of-care drug used in the treatment of malignant brain tumors. Furthermore, the IAG scaffold represents a new chemical space for the exploration of TLS pol inhibitors, which could prove useful as a strategy for improving patient response to genotoxic drugs.

Interaction of human SUV3 RNA/DNA helicase with BLM helicase; loss of the SUV3 gene results in mouse embryonic lethality.

The SUV3 gene is present in all eukaryotes and encodes an RNA/DNA helicase which operates both in mitochondria and cell nuclei. To assess its function in mammals we generated a mouse mutant strain in which the 3' part of the SUV3 gene is disrupted. The mutated allele is a hypomorph transmitted from one generation to another at a frequency about 35% lower than expected while mice homozygous for the mutation die in utero before midgestation. Using ELISA binding assays we show that human SUV3 protein interacts with human WRN and BLM helicases. The binding to BLM protein was 10-fold stronger (with a K(d) of 0.5nM) than to WRN protein (K(d) of 5nM). Silencing of the SUV3 gene in the human cell line HeLa resulted in elevation of homologous recombination as measured by the frequency of sister chromatid exchange during mitotic cell division. These results indicate that the SUV3 protein is required in mammalian development and in somatic cells participates in genome maintenance through interaction with other genome fidelity housekeepers.

Bacterial DNA repair genes and their eukaryotic homologues: 4. The role of nucleotide excision DNA repair (NER) system in mammalian cells.

The eukaryotic cell encounters more than one million various kinds of DNA lesions per day. The nucleotide excision repair (NER) pathway is one of the most important repair mechanisms that removes a wide spectrum of different DNA lesions. NER operates through two sub pathways: global genome repair (GGR) and transcription-coupled repair (TCR). GGR repairs the DNA damage throughout the entire genome and is initiated by the HR23B/XPC complex, while the CSB protein-governed TCR process removes DNA lesions from the actively transcribed strand. The sequence of events and the role of particular NER proteins are currently being extensively discussed. NER proteins also participate in other cellular processes like replication, transcription, chromatin maintenance and protein turnover. Defects in NER underlay severe genetic disorders: xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD).