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.

Site-Specific Synthesis of Oligonucleotides Containing 6-Oxo-M1dG, the Genomic Metabolite of M1dG, and Liquid Chromatography-Tandem Mass Spectrometry Analysis of Its In Vitro Bypass by Human Polymerase ι.

The lipid peroxidation product malondialdehyde and the DNA peroxidation product base-propenal react with dG to generate the exocyclic adduct, M1dG. This mutagenic lesion has been found in human genomic and mitochondrial DNA. M1dG in genomic DNA is enzymatically oxidized to 6-oxo-M1dG, a lesion of currently unknown mutagenic potential. Here, we report the synthesis of an oligonucleotide containing 6-oxo-M1dG and the results of extension experiments aimed at determining the effect of the 6-oxo-M1dG lesion on the activity of human polymerase iota (hPol ι). For this purpose, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed to obtain reliable quantitative data on the utilization of poorly incorporated nucleotides. Results demonstrate that hPol ι primarily incorporates deoxycytidine triphosphate (dCTP) and thymidine triphosphate (dTTP) across from 6-oxo-M1dG with approximately equal efficiency, whereas deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) are poor substrates. Following the incorporation of a single nucleotide opposite the lesion, 6-oxo-M1dG blocks further replication by the enzyme.

LC8/DYNLL1 is a 53BP1 effector and regulates checkpoint activation.

The tumor suppressor protein 53BP1 plays key roles in response to DNA double-strand breaks (DSBs) by serving as a master scaffold at the damaged chromatin. Current evidence indicates that 53BP1 assembles a cohort of DNA damage response (DDR) factors to distinctly execute its repertoire of DSB responses, including checkpoint activation and non-homologous end joining (NHEJ) repair. Here, we have uncovered LC8 (a.k.a. DYNLL1) as an important 53BP1 effector. We found that LC8 accumulates at laser-induced DNA damage tracks in a 53BP1-dependent manner and requires the canonical H2AX-MDC1-RNF8-RNF168 signal transduction cascade. Accordingly, genetic inactivation of LC8 or its interaction with 53BP1 resulted in checkpoint defects. Importantly, loss of LC8 alleviated the hypersensitivity of BRCA1-depleted cells to ionizing radiation and PARP inhibition, highlighting the 53BP1-LC8 module in counteracting BRCA1-dependent functions in the DDR. Together, these data establish LC8 as an important mediator of a subset of 53BP1-dependent DSB responses.

Biobanked Glioblastoma Patient-Derived Organoids as a Precision Medicine Model to Study Inhibition of Invasion.

Glioblastoma (GBM) is highly resistant to treatment and invasion into the surrounding brain is a cancer hallmark that leads to recurrence despite surgical resection. With the emergence of precision medicine, patient-derived 3D systems are considered potentially robust GBM preclinical models. In this study, we screened a library of 22 anti-invasive compounds (i.e., NF-kB, GSK-3-B, COX-2, and tubulin inhibitors) using glioblastoma U-251 MG cell spheroids. We evaluated toxicity and invasion inhibition using a 3D Matrigel invasion assay. We next selected three compounds that inhibited invasion and screened them in patient-derived glioblastoma organoids (GBOs). We developed a platform using available macros for FIJI/ImageJ to quantify invasion from the outer margin of organoids. Our data demonstrated that a high-throughput invasion screening can be done using both an established cell line and patient-derived 3D model systems. Tubulin inhibitor compounds had the best efficacy with U-251 MG cells, however, in ex vivo patient organoids the results were highly variable. Our results indicate that the efficacy of compounds is highly related to patient intra and inter-tumor heterogeneity. These results indicate that such models can be used to evaluate personal oncology therapeutic strategies.

A Facile Semisynthesis and Evaluation of Garcinoic Acid and Its Analogs for the Inhibition of Human DNA Polymerase ß.

Garcinoic acid has been identified as an inhibitor of DNA polymerase ß (pol ß). However, no structure-activity relationship (SAR) studies of garcinoic acid as a pol ß inhibitor have been conducted, in part due to the lack of an efficient synthetic method for this natural product and its analogs. We developed an efficient semi-synthetic method for garcinoic acid and its analogs by starting from natural product δ-tocotrienol. Our preliminary SAR studies provided a valuable insight into future discovery of garcinoic acid-based pol ß inhibitors.

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.

A catch and release program for single-stranded DNA.

Uncovering the mechanisms by which single-stranded binding proteins both protect and expose single-stranded DNA has important implications for our understanding of DNA replication and repair. A new study serves up a master class in developing a full kinetic model for one such protein, mtSSB, showing how DNA can be reeled in and set free to control accessibility.

Residues in the RecQ C-terminal Domain of the Human Werner Syndrome Helicase Are Involved in Unwinding G-quadruplex DNA.

The structural and biophysical properties typically associated with G-quadruplex (G4) structures render them a significant block for DNA replication, which must be overcome for cell division to occur. The Werner syndrome protein (WRN) is a RecQ family helicase that has been implicated in the efficient processing of G4 DNA structures. The aim of this study was to identify the residues of WRN involved in the binding and ATPase-driven unwinding of G4 DNA. Using a c-Myc G4 DNA model sequence and recombinant WRN, we have determined that the RecQ-C-terminal (RQC) domain of WRN imparts a 2-fold preference for binding to G4 DNA relative to non-G4 DNA substrates. NMR studies identified residues involved specifically in interactions with G4 DNA. Three of the amino acids in the WRN RQC domain that exhibited the largest G4-specific changes in NMR signal were then mutated alone or in combination. Mutating individual residues implicated in G4 binding had a modest effect on WRN binding to DNA, decreasing the preference for G4 substrates by ∼25%. Mutating two G4-interacting residues (T1024G and T1086G) abrogated preferential binding of WRN to G4 DNA. Very modest decreases in G4 DNA-stimulated ATPase activity were observed for the mutant enzymes. Most strikingly, G4 unwinding by WRN was inhibited ∼50% for all three point mutants and >90% for the WRN double mutant (T1024G/T1086G) relative to normal B-form dsDNA substrates. Our work has helped to identify residues in the WRN RQC domain that are involved specifically in the interaction with G4 DNA.

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.

Translesion DNA Synthesis in Cancer: Molecular Mechanisms and Therapeutic Opportunities.

The genomic landscape of cancer is one marred by instability, but the mechanisms that underlie these alterations are multifaceted and remain a topic of intense research. Cellular responses to DNA damage and/or replication stress can affect genome stability in tumors and influence the response of patients to therapy. In addition to direct repair, DNA damage tolerance (DDT) is an element of genomic maintenance programs that contributes to the etiology of several types of cancer. DDT mechanisms primarily act to resolve replication stress, and this can influence the effectiveness of genotoxic drugs. Translesion DNA synthesis (TLS) is an important component of DDT that facilitates direct bypass of DNA adducts and other barriers to replication. The central role of TLS in the bypass of drug-induced DNA lesions, the promotion of tumor heterogeneity, and the involvement of these enzymes in the maintenance of the cancer stem cell niche presents an opportunity to leverage inhibition of TLS as a way of improving existing therapies. In the review that follows, we summarize mechanisms of DDT, misregulation of TLS in cancer, and discuss the potential for targeting these pathways as a means of improving cancer therapies.

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.

Aberrant Kynurenine Signaling Modulates DNA Replication Stress Factors and Promotes Genomic Instability in Gliomas.

Metabolism of the essential amino acid L-tryptophan (TRP) is implicated in a number of neurological conditions including depression, neurodegenerative diseases, and cancer. The TRP catabolite kynurenine (KYN) has recently emerged as an important neuroactive factor in brain tumor pathogenesis, with additional studies implicating KYN in other types of cancer. Often highlighted as a modulator of the immune response and a contributor to immune escape for malignant tumors, it is well-known that KYN has effects on the production of the coenzyme nicotinamide adenine dinucleotide (NAD(+)), which can have a direct impact on DNA repair, replication, cell division, redox signaling, and mitochondrial function. Additional effects of KYN signaling are imparted through its role as an endogenous agonist for the aryl hydrocarbon receptor (AhR), and it is largely through activation of the AhR that KYN appears to mediate malignant progression in gliomas. We have recently reported on the ability of KYN signaling to modulate expression of human DNA polymerase kappa (hpol κ), a translesion enzyme involved in bypass of bulky DNA lesions and activation of the replication stress response. Given the impact of KYN on NAD(+) production, AhR signaling, and translesion DNA synthesis, it follows that dysregulation of KYN signaling in cancer may promote malignancy through alterations in the level of endogenous DNA damage and replication stress. In this perspective, we discuss the connections between KYN signaling, DNA damage tolerance, and genomic instability, as they relate to cancer.

Effects of Twelve Germline Missense Variations on DNA Lesion and G-Quadruplex Bypass Activities of Human DNA Polymerase REV1.

The Y-family DNA polymerase REV1 is involved in replicative bypass of damaged DNA and G-quadruplex (G4) DNA. In addition to a scaffolding role in the replicative bypass, REV1 acts in a catalytic role as a deoxycytidyl transferase opposite some replication stall sites, e.g., apurinic/apyrimidinic (AP) sites, N(2)-guanyl lesions, and G4 sites. We characterized the biochemical properties of 12 reported germline missense variants of human REV1, including the N373S variant associated with high risk of cervical cancer, using the recombinant REV1 (residues 330-833) proteins and DNA templates containing a G, AP site, N(2)-CH2(2-naphthyl)G (N(2)-NaphG), or G4. In steady-state kinetic analyses, the F427L, R434Q, M656V, D700N, R704Q, and P831L variants displayed 2- to 8-fold decreases in kcat/Km for dCTP insertion opposite all four templates, compared to that of wild-type, while the N373S, M407L, and N497S showed 2- to 3-fold increases with all four and the former three or two templates, respectively. The F427L, R434Q, M656V, and R704Q variants also had 2- to 3-fold lower binding affinities to DNA substrates containing G, an AP site, and/or N(2)-NaphG than wild-type. Distinctively, the N373S variant had a 3-fold higher binding affinity to G4 DNA than the wild-type, as well as a 2-fold higher catalytic activity opposite the first tetrad G, suggesting a facilitating effect of this variation on replication of G4 DNA sequences in certain human papillomavirus genomes. Our results suggest that the catalytic function of REV1 is moderately or slightly altered by at least nine genetic variations, and the G4 DNA processing function of REV1 is slightly enhanced by the N373S variation, which might provide the possibility that certain germline missense REV1 variations affect the individual susceptibility to carcinogenesis by modifying the capability of REV1 for replicative bypass past DNA lesions and G4 motifs derived from chemical and viral carcinogens.

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.

Antinociceptive effects of the 6-O-sulfate ester of morphine in normal and diabetic rats: Comparative role of mu- and delta-opioid receptors.

This study determined the antinociceptive effects of morphine and morphine-6-O-sulfate (M6S) in both normal and diabetic rats, and evaluated the comparative role of mu-opioid receptors (mu-ORs) and delta-opioid receptors (delta-ORs) in the antinociceptive action of these opioids. In vitro characterization of mu-OR and delta-OR-mediated signaling by M6S and morphine in stably transfected Chinese hamster ovary (CHO-K1) cells showed that M6S exhibited a 6-fold higher affinity for delta-ORs and modulated G-protein and adenylyl cyclase activity via delta-ORs more potently than morphine. Interestingly, while morphine acted as a full agonist at delta-ORs in both functional assays examined, M6S exhibited either partial or full agonist activity for modulation of G-protein or adenylyl cyclase activity, respectively. Molecular docking studies indicated that M6S but not morphine binds equally well at the ligand binding site of both mu- and delta-ORs. In vivo analgesic effects of M6S and morphine in both normal and streptozotocin-induced diabetic Sprague-Dawley rats utilizing the hot water tail flick latency test showed that M6S produced more potent antinociception than morphine in both normal rats and diabetic rats. This difference in potency was abrogated following antagonism of delta- but not mu- or kappa (kappa-ORs) opioid receptors. During 9days of chronic treatment, tolerance developed to morphine-treated but not to M6S-treated rats. Rats that developed tolerance to morphine still remained responsive to M6S. Collectively, this study demonstrates that M6S is a potent and efficacious mu/delta opioid analgesic with a delayed tolerance profile when compared to morphine in both normal and diabetic rats. PERSPECTIVE: This study demonstrates that M6S acts at both mu- and delta-ORs, and adds to the growing evidence that the use of mixed mu/delta opioid agonists in pain treatment may have clinical benefit.

Dioxol and dihydrodioxin analogs of 2- and 3-phenylacetonitriles as potent anti-cancer agents with nanomolar activity against a variety of human cancer cells.

A small library of (Z)-2-(benzo[d][1,3]dioxol-5-yl) and (Z)-2,3-dihydrobenzo[b][1,4]dioxin-6-yl analogs of 2- and 3-phenylacetonitriles has been synthesized and evaluated for their anti-cancer activities against a panel of 60 human cancer cell lines. The dihydrodioxin analog 3j and dioxol analogs 5e and 7e exhibited the most potent anti-cancer activity of all the analogs synthesized in this study, with GI50 values of <100 nM against almost all of the cell lines in the human cancer cell panel. Of these three, only compound 3j inhibited tubulin polymerization to any degree in vitro. The binding modes of 3j and the structurally related tubulin-inhibitor DMU-212 were determined by virtual docking studies with tubulin dimer. Compound 3j docked at the colchicine-binding site at the dimer interface of tubulin. The Full-Fitness (FF) score of 3j was observed to be substantially higher than DMU-212, which agrees well with the observed anti-cancer potency (GI50 values). The mechanism by which dioxol analogs 5e and 7e exert their cytotoxic effects remains unknown at this stage, but it is unlikely that they affect tubulin dynamics. Nevertheless, these findings suggest that both dioxol and dihydrodioxin analogs of phenylacrylonitrile may have potential for development as clinical candidates to treat a variety of human cancers.

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.

Mammalian Exo1 encodes both structural and catalytic functions that play distinct roles in essential biological processes.

Mammalian Exonuclease 1 (EXO1) is an evolutionarily conserved, multifunctional exonuclease involved in DNA damage repair, replication, immunoglobulin diversity, meiosis, and telomere maintenance. It has been assumed that EXO1 participates in these processes primarily through its exonuclease activity, but recent studies also suggest that EXO1 has a structural function in the assembly of higher-order protein complexes. To dissect the enzymatic and nonenzymatic roles of EXO1 in the different biological processes in vivo, we generated an EXO1-E109K knockin (Exo1(EK)) mouse expressing a stable exonuclease-deficient protein and, for comparison, a fully EXO1-deficient (Exo1(null)) mouse. In contrast to Exo1(null/null) mice, Exo1(EK/EK) mice retained mismatch repair activity and displayed normal class switch recombination and meiosis. However, both Exo1-mutant lines showed defects in DNA damage response including DNA double-strand break repair (DSBR) through DNA end resection, chromosomal stability, and tumor suppression, indicating that the enzymatic function is required for those processes. On a transformation-related protein 53 (Trp53)-null background, the DSBR defect caused by the E109K mutation altered the tumor spectrum but did not affect the overall survival as compared with p53-Exo1(null) mice, whose defects in both DSBR and mismatch repair also compromised survival. The separation of these functions demonstrates the differential requirement for the structural function and nuclease activity of mammalian EXO1 in distinct DNA repair processes and tumorigenesis in vivo.

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.