Hypoxia-dependent recruitment of error-prone DNA polymerases to genome replication.

Hypoxia is common in tumors and is associated with cancer progression and drug resistance, driven, at least in part, by genetic instability. Little is known on how hypoxia affects Translesion DNA Synthesis (TLS), in which error-prone DNA polymerases bypass lesions, thereby maintaining DNA continuity at the price of increased mutations. Here we show that under acute hypoxia, PCNA monoubiquitination, a key step in TLS, and expression of error-prone DNA polymerases increased under regulation of the HIF1α transcription factor. Knocking-down expression of DNA polymerase η, or using PCNA ubiquitination-resistant cells, inhibited genomic DNA replication specifically under hypoxia, and iPOND analysis revealed massive recruitment of TLS DNA polymerases to nascent DNA under hypoxia, uncovering a dramatic involvement of error-prone DNA polymerases in genomic replication. Of note, expression of TLS-polymerases correlates with VEGFA (primary HIF1α target) in a database of renal cell carcinoma, a cancer which accumulates HIF1α. Our results suggest that the tumor microenvironment can lead the cell to forgo, to some extent, the fast and accurate canonical DNA polymerases, for the more flexible and robust, but low-fidelity TLS DNA polymerases. This might endow cancer cells with resilience to overcome replication stress, and mutability to escape the immune system and chemotherapeutic drugs.

A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring Tools.

Personalized vaccines against patient-unique tumor-associated antigens represent a promising new approach for cancer immunotherapy. Vaccine efficacy is assessed by quantification of changes in the frequency and/or the activity of antigen-specific T cells. Enzyme-linked immunosorbent spot (ELISpot) and flow cytometry (FCM) are methodologies frequently used for assessing vaccine efficacy. We tested these methodologies and found that both ELISpot and standard FCM [monitoring CD3/CD4/CD8/IFNγ/Viability+CD14+CD19 (dump)] demonstrate background IFNγ secretion, which, in many cases, was higher than the antigen-specific signal measured by the respective methodology (frequently ranging around 0.05-0.2%). To detect such weak T-cell responses, we developed an FCM panel that included two early activation markers, 4-1BB (CD137) and CD40L (CD154), in addition to the above-cited markers. These two activation markers have a close to zero background expression and are rapidly upregulated following antigen-specific activation. They enabled the quantification of rare T cells responding to antigens within the assay well. Background IFNγ-positive CD4 T cell frequencies decreased to 0.019% ± 0.028% and CD8 T cells to 0.009% ± 0.013%, which are 19 and 13 times lower, respectively, than without the use of these markers. The presented methodology enables highly sensitive monitoring of T-cell responses to tumor-associated antigens in the very low, but clinically relevant, frequencies.

Effective bubble-based testing for SARS-CoV-2 using swab-pooling.

OBJECTIVES: Despite the success in developing COVID-19 vaccines, containment of the disease is obstructed worldwide by vaccine production bottlenecks, logistics hurdles, vaccine refusal, transmission through unvaccinated children, and the appearance of new viral variants. This underscores the need for effective strategies for identifying carriers/patients, which was the main aim of this study. METHODS: We present a bubble-based PCR testing approach using swab-pooling into lysis buffer. A bubble is a cluster of people who can be periodically tested for SARS-CoV-2 by swab-pooling. A positive test of a pool mandates quarantining each of its members, who are then individually tested while in isolation to identify the carrier(s) for further epidemiological contact tracing. RESULTS: We tested an overall sample of 25 831 individuals, divided into 1273 bubbles, with an average size of 20.3 ± 7.7 swabs/test tube, obtaining for all pools (≤37 swabs/pool) a specificity of 97.5% (lower bound 96.6%) and a sensitivity of 86.3% (lower bound 78.2%) and a post hoc analyzed sensitivity of 94.6% (lower bound 86.7%) and a specificity of 97.2% (lower bound 96.2%) in pools with ≤25 swabs, relative to individual testing. DISCUSSION: This approach offers a significant scale-up in sampling and testing throughput and savings in testing cost, without reducing sensitivity or affecting the standard PCR testing laboratory routine. It can be used in school classes, airplanes, hospitals, military units, and workplaces, and may be applicable to future pandemics.

TENT4A Non-Canonical Poly(A) Polymerase Regulates DNA-Damage Tolerance via Multiple Pathways That Are Mutated in Endometrial Cancer.

TENT4A (PAPD7) is a non-canonical poly(A) polymerase, of which little is known. Here, we show that TENT4A regulates multiple biological pathways and focuses on its multilayer regulation of translesion DNA synthesis (TLS), in which error-prone DNA polymerases bypass unrepaired DNA lesions. We show that TENT4A regulates mRNA stability and/or translation of DNA polymerase η and RAD18 E3 ligase, which guides the polymerase to replication stalling sites and monoubiquitinates PCNA, thereby enabling recruitment of error-prone DNA polymerases to damaged DNA sites. Remarkably, in addition to the effect on RAD18 mRNA stability via controlling its poly(A) tail, TENT4A indirectly regulates RAD18 via the tumor suppressor CYLD and via the long non-coding antisense RNA PAXIP1-AS2, which had no known function. Knocking down the expression of TENT4A or CYLD, or overexpression of PAXIP1-AS2 led each to reduced amounts of the RAD18 protein and DNA polymerase η, leading to reduced TLS, highlighting PAXIP1-AS2 as a new TLS regulator. Bioinformatics analysis revealed that TLS error-prone DNA polymerase genes and their TENT4A-related regulators are frequently mutated in endometrial cancer genomes, suggesting that TLS is dysregulated in this cancer.

Recurrent deletions in clonal hematopoiesis are driven by microhomology-mediated end joining.

The mutational mechanisms underlying recurrent deletions in clonal hematopoiesis are not entirely clear. In the current study we inspect the genomic regions around recurrent deletions in myeloid malignancies, and identify microhomology-based signatures in CALR, ASXL1 and SRSF2 loci. We demonstrate that these deletions are the result of double stand break repair by a PARP1 dependent microhomology-mediated end joining (MMEJ) pathway. Importantly, we provide evidence that these recurrent deletions originate in pre-leukemic stem cells. While DNA polymerase theta (POLQ) is considered a key component in MMEJ repair, we provide evidence that pre-leukemic MMEJ (preL-MMEJ) deletions can be generated in POLQ knockout cells. In contrast, aphidicolin (an inhibitor of replicative polymerases and replication) treatment resulted in a significant reduction in preL-MMEJ. Altogether, our data indicate an association between POLQ independent MMEJ and clonal hematopoiesis and elucidate mutational mechanisms involved in the very first steps of leukemia evolution.

Improved sensitivity, safety, and rapidity of COVID-19 tests by replacing viral storage solution with lysis buffer.

Conducting numerous, rapid, and reliable PCR tests for SARS-CoV-2 is essential for our ability to monitor and control the current COVID-19 pandemic. Here, we tested the sensitivity and efficiency of SARS-CoV-2 detection in clinical samples collected directly into a mix of lysis buffer and RNA preservative, thus inactivating the virus immediately after sampling. We tested 79 COVID-19 patients and 20 healthy controls. We collected two samples (nasopharyngeal swabs) from each participant: one swab was inserted into a test tube with Viral Transport Medium (VTM), following the standard guideline used as the recommended method for sample collection; the other swab was inserted into a lysis buffer supplemented with nucleic acid stabilization mix (coined NSLB). We found that RT-qPCR tests of patients were significantly more sensitive with NSLB sampling, reaching detection threshold 2.1±0.6 (Mean±SE) PCR cycles earlier then VTM samples from the same patient. We show that this improvement is most likely since NSLB samples are not diluted in lysis buffer before RNA extraction. Re-extracting RNA from NSLB samples after 72 hours at room temperature did not affect the sensitivity of detection, demonstrating that NSLB allows for long periods of sample preservation without special cooling equipment. We also show that swirling the swab in NSLB and discarding it did not reduce sensitivity compared to retaining the swab in the tube, thus allowing improved automation of COVID-19 tests. Overall, we show that using NSLB instead of VTM can improve the sensitivity, safety, and rapidity of COVID-19 tests at a time most needed.

Multi-center nationwide comparison of seven serology assays reveals a SARS-CoV-2 non-responding seronegative subpopulation.

BACKGROUND: An Israeli national taskforce performed a multi-center clinical and analytical validation of seven serology assays to determine their utility and limitations for SARS-CoV-2 diagnosis. METHODS: Serology assays from Roche, Abbott, Diasorin, BioMerieux, Beckman-Coulter, Siemens, and an in-house RBD ELISA were included. Negative samples from 2391 individuals representative of the Israeli population, and 698 SARS-CoV-2 PCR positive patients, collected between March and May 2020, were analyzed. FINDINGS: Immunoassays sensitivities between 81.5%-89.4% and specificities between 97.7%-100% resulted in a profound impact on the expected Positive Predictive Value (PPV) in low (<15%) prevalence scenarios. No meaningful increase was detected in the false positive rate in children compared to adults. A positive correlation between disease severity and antibody titers, and no decrease in antibody titers in the first 8 weeks after PCR positivity was observed. We identified a subgroup of symptomatic SARS-CoV-2 positive patients (~5% of patients), who remained seronegative across a wide range of antigens, isotypes, and technologies. INTERPRETATION: The commercially available automated immunoassays exhibit significant differences in performance and expected PPV in low prevalence scenarios. The low false-positivity rate in under 20's suggests that cross-reactive immunity from previous CoV strains is unlikely to explain the milder disease course in children. Finding no decrease in antibody titers in the first 8 weeks is in contrast to some reports of short half-life for SARS-CoV-2 antibodies. The ~5% who were seronegative non-responders, using multiple assays in a population-wide manner, represents the proportion of patients that may be at risk for re-infection. FUNDING: Israel Ministry of Health.

DNA Repair Biomarker for Lung Cancer Risk and its Correlation With Airway Cells Gene Expression.

BACKGROUND: Improving lung cancer risk assessment is required because current early-detection screening criteria miss most cases. We therefore examined the utility for lung cancer risk assessment of a DNA Repair score obtained from OGG1, MPG, and APE1 blood tests. In addition, we examined the relationship between the level of DNA repair and global gene expression. METHODS: We conducted a blinded case-control study with 150 non-small cell lung cancer case patients and 143 control individuals. DNA Repair activity was measured in peripheral blood mononuclear cells, and the transcriptome of nasal and bronchial cells was determined by RNA sequencing. A combined DNA Repair score was formed using logistic regression, and its correlation with disease was assessed using cross-validation; correlation of expression to DNA Repair was analyzed using Gene Ontology enrichment. RESULTS: DNA Repair score was lower in case patients than in control individuals, regardless of the case's disease stage. Individuals at the lowest tertile of DNA Repair score had an increased risk of lung cancer compared to individuals at the highest tertile, with an odds ratio (OR) of 7.2 (95% confidence interval [CI] = 3.0 to 17.5; P < .001), and independent of smoking. Receiver operating characteristic analysis yielded an area under the curve of 0.89 (95% CI = 0.82 to 0.93). Remarkably, low DNA Repair score correlated with a broad upregulation of gene expression of immune pathways in patients but not in control individuals. CONCLUSIONS: The DNA Repair score, previously shown to be a lung cancer risk factor in the Israeli population, was validated in this independent study as a mechanism-based cancer risk biomarker and can substantially improve current lung cancer risk prediction, assisting prevention and early detection by computed tomography scanning.

Mutational signatures reveal the role of RAD52 in p53-independent p21-driven genomic instability.

BACKGROUND: Genomic instability promotes evolution and heterogeneity of tumors. Unraveling its mechanistic basis is essential for the design of appropriate therapeutic strategies. In a previous study, we reported an unexpected oncogenic property of p21WAF1/Cip1, showing that its chronic expression in a p53-deficient environment causes genomic instability by deregulation of the replication licensing machinery. RESULTS: We now demonstrate that p21WAF1/Cip1 can further fuel genomic instability by suppressing the repair capacity of low- and high-fidelity pathways that deal with nucleotide abnormalities. Consequently, fewer single nucleotide substitutions (SNSs) occur, while formation of highly deleterious DNA double-strand breaks (DSBs) is enhanced, crafting a characteristic mutational signature landscape. Guided by the mutational signatures formed, we find that the DSBs are repaired by Rad52-dependent break-induced replication (BIR) and single-strand annealing (SSA) repair pathways. Conversely, the error-free synthesis-dependent strand annealing (SDSA) repair route is deficient. Surprisingly, Rad52 is activated transcriptionally in an E2F1-dependent manner, rather than post-translationally as is common for DNA repair factor activation. CONCLUSIONS: Our results signify the importance of mutational signatures as guides to disclose the repair history leading to genomic instability. We unveil how chronic p21WAF1/Cip1 expression rewires the repair process and identifies Rad52 as a source of genomic instability and a candidate therapeutic target.

High-resolution genomic assays provide insight into the division of labor between TLS and HDR in mammalian replication of damaged DNA.

The multitude of DNA lesions that continuously form in DNA cannot all be detected and removed prior to replication. Thus, encounters of the replication fork with DNA damage become inevitable. Such encounters inhibit fork progression, leading to replication fork arrest or to replication re-priming downstream of the damage site. Either of these events will result in the formation of gap-lesion structures, in which a damaged base is located in a single stranded stretch of DNA, that is vulnerable to subsequent nicking. The double strand break that would ensue if ssDNA becomes nicked constitutes escalation of the damage from nucleotide(s)-specific to chromosomal scale. Cells employ two universal DNA damage tolerance (DDT) strategies to resolve these situations, by converting the gap-lesion structures into dsDNA without repairing the damage. The first is translesion DNA synthesis (TLS), in which a specialized low-fidelity DNA polymerase inserts a nucleotide opposite the damaged one. TLS is inherently mutagenic, due to the miscoding nature of most damaged nucleotides. The second strategy is homology-dependent repair (HDR), which relies on the presence of an identical intact sister chromatid. The molecular mechanisms that regulate the division of labor between these pathways are poorly understood. This review focuses on the balance between TLS and HDR in mammalian cells, discussing recent findings that were made possible thanks to newly developed high resolution genomic assays, and highlighting the role of the DNA lesion's properties in DDT pathway choice.

In vivo evidence for translesion synthesis by the replicative DNA polymerase δ.

The intolerance of DNA polymerase δ (Polδ) to incorrect base pairing contributes to its extremely high accuracy during replication, but is believed to inhibit translesion synthesis (TLS). However, chicken DT40 cells lacking the POLD3 subunit of Polδ are deficient in TLS. Previous genetic and biochemical analysis showed that POLD3 may promote lesion bypass by Polδ itself independently of the translesion polymerase Polζ of which POLD3 is also a subunit. To test this hypothesis, we have inactivated Polδ proofreading in pold3 cells. This significantly restored TLS in pold3 mutants, enhancing dA incorporation opposite abasic sites. Purified proofreading-deficient human Polδ holoenzyme performs TLS of abasic sites in vitro much more efficiently than the wild type enzyme, with over 90% of TLS events resulting in dA incorporation. Furthermore, proofreading deficiency enhances the capability of Polδ to continue DNA synthesis over UV lesions both in vivo and in vitro These data support Polδ contributing to TLS in vivo and suggest that the mutagenesis resulting from loss of Polδ proofreading activity may in part be explained by enhanced lesion bypass.

DNA sequence context greatly affects the accuracy of bypass across an ultraviolet light 6-4 photoproduct in mammalian cells.

Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism carried out by low-fidelity DNA polymerases that bypass DNA lesions, which overcomes replication stalling. Despite the miscoding nature of most common DNA lesions, several of them are bypassed in mammalian cells in a relatively accurate manner, which plays a key role maintaining a low mutation load. Whereas it is generally agreed that TLS across the major UV and sunlight induced DNA lesion, the cyclobutane pyrimidine dimer (CPD), is accurate, there were conflicting reports on whether the same is true for the thymine-thymine pyrimidine-pyrimidone(6-4) ultraviolet light photoproduct (TT6-4PP), which represents the second most common class of UV lesions. Using a TLS assay system based on gapped plasmids carrying site-specific TT6-4PP lesions in defined sequence contexts we show that the DNA sequence context markedly affected both the extent and accuracy of TLS. The sequence exhibiting higher TLS exhibited also higher error-frequency, caused primarily by semi-targeted mutations, at the nearest nucleotides flanking the lesion. Our results resolve the discrepancy reported on TLS across TT6-4PP, and suggest that TLS is more accurate in human cells than in mouse cells.

Development of APE1 enzymatic DNA repair assays: low APE1 activity is associated with increase lung cancer risk.

The key role of DNA repair in removing DNA damage and minimizing mutations makes it an attractive target for cancer risk assessment and prevention. Here we describe the development of a robust assay for apurinic/apyrimidinic (AP) endonuclease 1 (APE1; APEX1), an essential enzyme involved in the repair of oxidative DNA damage. APE1 DNA repair enzymatic activity was measured in peripheral blood mononuclear cell protein extracts using a radioactivity-based assay, and its association with lung cancer was determined using conditional logistic regression with specimens from a population-based case-control study with 96 lung cancer cases and 96 matched control subjects. The mean APE1 enzyme activity in case patients was 691 [95% confidence interval (CI) = 655-727] units/ng protein, significantly lower than in control subjects (mean = 793, 95% CI = 751-834 units/ng protein, P = 0.0006). The adjusted odds ratio for lung cancer associated with 1 SD (211 units) decrease in APE1 activity was 2.0 (95% CI = 1.3-3.1; P = 0.002). Comparison of radioactivity- and fluorescence-based assays showed that the two are equivalent, indicating no interference by the fluorescent tag. The APE1Asp148Glu SNP was associated neither with APE1 enzyme activity nor with lung cancer risk. Taken together, our results indicate that low APE1 activity is associated with lung cancer risk, consistent with the hypothesis that 'bad DNA repair', rather than 'bad luck', is involved in cancer etiology. Such assays may be useful, along with additional DNA repair biomarkers, for risk assessment of lung cancer and perhaps other cancers, and for selecting individuals to undergo early detection techniques such as low-dose CT.

DNA lesion identity drives choice of damage tolerance pathway in murine cell chromosomes.

DNA-damage tolerance (DDT) via translesion DNA synthesis (TLS) or homology-dependent repair (HDR) functions to bypass DNA lesions encountered during replication, and is critical for maintaining genome stability. Here, we present piggyBlock, a new chromosomal assay that, using piggyBac transposition of DNA containing a known lesion, measures the division of labor between the two DDT pathways. We show that in the absence of DNA damage response, tolerance of the most common sunlight-induced DNA lesion, TT-CPD, is achieved by TLS in mouse embryo fibroblasts. Meanwhile, BP-G, a major smoke-induced DNA lesion, is bypassed primarily by HDR, providing the first evidence for this mechanism being the main tolerance pathway for a biologically important lesion in a mammalian genome. We also show that, far from being a last-resort strategy as it is sometimes portrayed, TLS operates alongside nucleotide excision repair, handling 40% of TT-CPDs in repair-proficient cells. Finally, DDT acts in mouse embryonic stem cells, exhibiting the same pattern­mutagenic TLS included­despite the risk of propagating mutations along all cell lineages. The new method highlights the importance of HDR, and provides an effective tool for studying DDT in mammalian cells.

Identification of novel DNA-damage tolerance genes reveals regulation of translesion DNA synthesis by nucleophosmin.

Cells cope with replication-blocking lesions via translesion DNA synthesis (TLS). TLS is carried out by low-fidelity DNA polymerases that replicate across lesions, thereby preventing genome instability at the cost of increased point mutations. Here we perform a two-stage siRNA-based functional screen for mammalian TLS genes and identify 17 validated TLS genes. One of the genes, NPM1, is frequently mutated in acute myeloid leukaemia (AML). We show that NPM1 (nucleophosmin) regulates TLS via interaction with the catalytic core of DNA polymerase-η (polη), and that NPM1 deficiency causes a TLS defect due to proteasomal degradation of polη. Moreover, the prevalent NPM1c+ mutation that causes NPM1 mislocalization in ~30% of AML patients results in excessive degradation of polη. These results establish the role of NPM1 as a key TLS regulator, and suggest a mechanism for the better prognosis of AML patients carrying mutations in NPM1.

Enzymatic MPG DNA repair assays for two different oxidative DNA lesions reveal associations with increased lung cancer risk.

DNA repair is a major mechanism for minimizing mutations and reducing cancer risk. Here, we present the development of reproducible and specific enzymatic assays for methylpurine DNA glycosylase (MPG) repairing the oxidative lesions 1,N6-ethenoadenine (εA) and hypoxanthine (Hx) in peripheral blood mononuclear cells protein extracts. Association of these DNA repair activities with lung cancer was determined using conditional logistic regression with specimens from a population-based case-control study with 96 lung cancer cases and 96 matched control subjects. The mean MPG-εA in case patients was 15.8 units/µg protein (95% CI 15.3-16.3), significantly higher than in control subjects-15.1 (14.6-15.5), *P = 0.011. The adjusted odds ratio for lung cancer associated with a one SD increase in MPG-εA activity (2.48 units) was significantly bigger than 1 (OR = 1.6, 95% CI = 1.1-2.4; *P = 0.013). When activity of OGG1, a different DNA repair enzyme for oxidative damage, was included in the model, the estimated odds ratio/SD for a combined MPG-εA-OGG1 score was 2.6 (95% CI 1.6-4.2) *P = 0.0001, higher than the odds ratio for each single assay. The MPG enzyme activity assays described provide robust functional risk biomarkers, with increased MPG-εA activity being associated with increased lung cancer risk, similar to the behavior of MPG-Hx. This underscores the notion that imbalances in DNA repair, including high DNA repair, usually perceived as beneficial, can cause cancer risk. Such DNA repair risk biomarkers may be useful for risk assessment of lung cancer and perhaps other cancer types, and for early detection techniques such as low-dose CT.

Low integrated DNA repair score and lung cancer risk.

DNA repair is a prime mechanism for preventing DNA damage, mutation, and cancers. Adopting a functional approach, we examined the association with lung cancer risk of an integrated DNA repair score, measured by a panel of three enzymatic DNA repair activities in peripheral blood mononuclear cells. The panel included assays for AP endonuclease 1 (APE1), 8-oxoguanine DNA glycosylase (OGG1), and methylpurine DNA glycosylase (MPG), all of which repair oxidative DNA damage as part of the base excision repair pathways. A blinded population-based case-control study was conducted with 96 patients with lung cancer and 96 control subjects matched by gender, age (±1 year), place of residence, and ethnic group (Jews/non-Jews). The three DNA repair activities were measured, and an integrated DNA repair OMA (OGG1, MPG, and APE1) score was calculated for each individual. Conditional logistic regression analysis revealed that individuals in the lowest tertile of the integrated DNA repair OMA score had an increased risk of lung cancer compared with the highest tertile, with OR = 9.7; 95% confidence interval (CI), 3.1-29.8; P < 0.001, or OR = 5.6; 95% CI, 2.1-15.1; P < 0.001 after cross-validation. These results suggest that pending validation, this DNA repair panel of risk factors may be useful for lung cancer risk assessment, assisting prevention and referral to early detection by technologies such as low-dose computed tomography scanning.

Genomic assay reveals tolerance of DNA damage by both translesion DNA synthesis and homology-dependent repair in mammalian cells.

DNA lesions can block replication forks and lead to the formation of single-stranded gaps. These replication complications are mitigated by DNA damage tolerance mechanisms, which prevent deleterious outcomes such as cell death, genomic instability, and carcinogenesis. The two main tolerance strategies are translesion DNA synthesis (TLS), in which low-fidelity DNA polymerases bypass the blocking lesion, and homology-dependent repair (HDR; postreplication repair), which is based on the homologous sister chromatid. Here we describe a unique high-resolution method for the simultaneous analysis of TLS and HDR across defined DNA lesions in mammalian genomes. The method is based on insertion of plasmids carrying defined site-specific DNA lesions into mammalian chromosomes, using phage integrase-mediated integration. Using this method we show that mammalian cells use HDR to tolerate DNA damage in their genome. Moreover, analysis of the tolerance of the UV light-induced 6-4 photoproduct, the tobacco smoke-induced benzo[a]pyrene-guanine adduct, and an artificial trimethylene insert shows that each of these three lesions is tolerated by both TLS and HDR. We also determined the specificity of nucleotide insertion opposite these lesions during TLS in human genomes. This unique method will be useful in elucidating the mechanism of DNA damage tolerance in mammalian chromosomes and their connection to pathological processes such as carcinogenesis.

N-methylpurine DNA glycosylase and OGG1 DNA repair activities: opposite associations with lung cancer risk.

Only a minority of smokers develop lung cancer, possibly due to genetic predisposition, including DNA repair deficiencies. To examine whether inter-individual variations in DNA repair activity of N-methylpurine DNA glycosylase (MPG) are associated with lung cancer, we conducted a blinded, population-based, case-control study with 100 lung cancer case patients and 100 matched control subjects and analyzed the data with conditional logistic regression. All statistical tests were two-sided. MPG enzyme activity in peripheral blood mononuclear cells from case patients was higher than in control subjects, results opposite that of 8-oxoguanine DNA glycosylase (OGG1) DNA repair enzyme activity. For lung cancer associated with one standard deviation increase in MPG activity, the adjusted odds ratio was 1.8 (95% confidence interval [CI] = 1.2 to 2.6; P = .006). A combined MPG and OGG1 activities score was more strongly associated with lung cancer risk than either activity alone, with an odds ratio of 2.3 (95% CI = 1.4 to 3.6; P < .001). These results form a basis for a future panel of risk biomarkers for lung cancer risk assessment and prevention.