Tracking break-induced replication shows that it stalls at roadblocks.

Break-induced replication (BIR) repairs one-ended double-strand breaks in DNA similar to those formed by replication collapse or telomere erosion, and it has been implicated in the initiation of genome instability in cancer and other human diseases1,2. Previous studies have defined the enzymes that are required for BIR1-5; however, understanding of initial and extended BIR synthesis, and of how the migrating D-loop proceeds through known replication roadblocks, has been precluded by technical limitations. Here we use a newly developed assay to show that BIR synthesis initiates soon after strand invasion and proceeds more slowly than S-phase replication. Without primase, leading strand synthesis is initiated efficiently, but is unable to proceed beyond 30 kilobases, suggesting that primase is needed for stabilization of the nascent leading strand. DNA synthesis can initiate in the absence of Pif1 or Pol32, but does not proceed efficiently. Interstitial telomeric DNA disrupts and terminates BIR progression, and BIR initiation is suppressed by transcription proportionally to the transcription level. Collisions between BIR and transcription lead to mutagenesis and chromosome rearrangements at levels that exceed instabilities induced by transcription during normal replication. Together, these results provide fundamental insights into the mechanism of BIR and how BIR contributes to genome instability.

ATM Inhibitor Suppresses Gemcitabine-Resistant BTC Growth in a Polymerase θ Deficiency-Dependent Manner.

Patients with advanced biliary tract cancer (BTC) inevitably experience progression after first-line, gemcitabine-based chemotherapy, due to chemo-resistance. The genetic alterations of DNA damage repair (DDR) genes are usually determined in BTC tumors. In this study, we found that the POLQ mRNA levels are downregulated and the ataxia-telangiectasia mutated (ATM) inhibitor AZD0156 was more sensitive in gemcitabine-resistant BTC sublines than in the parental cell lines. The knockdown of DNA polymerase θ does not affect cell proliferation, but its combination with the ATM inhibitor facilitated cell death in gemcitabine-resistant and gemcitabine-intensive BTC cells. Moreover, in the DNA damage caused by photon, hydrogen peroxide, or chemotherapy drugs, synthetic lethal interactions were found in combination with ATM inhibition by AZD0156 and DNA polymerase θ depletion, resulting in increased DNA damage accumulation and micronucleus formation, as well as reduced cell survival and colony formation. Collectively, our results reveal that ATM acts as a potential target in gemcitabine-resistant and DNA polymerase θ-deficient BTC.

Large deletions in immunoglobulin genes are associated with a sustained absence of DNA Polymerase η.

Somatic hypermutation of immunoglobulin genes is a highly mutagenic process that is B cell-specific and occurs during antigen-driven responses leading to antigen specificity and antibody affinity maturation. Mutations at the Ig locus are initiated by Activation-Induced cytidine Deaminase and are equally distributed at G/C and A/T bases. This requires the establishment of error-prone repair pathways involving the activity of several low fidelity DNA polymerases. In the physiological context, the G/C base pair mutations involve multiple error-prone DNA polymerases, while the generation of mutations at A/T base pairs depends exclusively on the activity of DNA polymerase η. Using two large cohorts of individuals with xeroderma pigmentosum variant (XP-V), we report that the pattern of mutations at Ig genes becomes highly enriched with large deletions. This observation is more striking for patients older than 50 years. We propose that the absence of Pol η allows the recruitment of other DNA polymerases that profoundly affect the Ig genomic landscape.

DNA polymerase ζ in DNA replication and repair.

Here, we survey the diverse functions of DNA polymerase ζ (pol ζ) in eukaryotes. In mammalian cells, REV3L (3130 residues) is the largest catalytic subunit of the DNA polymerases. The orthologous subunit in yeast is Rev3p. Pol ζ also includes REV7 subunits (encoded by Rev7 in yeast and MAD2L2 in mammalian cells) and two subunits shared with the replicative DNA polymerase, pol δ. Pol ζ is used in response to circumstances that stall DNA replication forks in both yeast and mammalian cells. The best-examined situation is translesion synthesis at sites of covalent DNA lesions such as UV radiation-induced photoproducts. We also highlight recent evidence that uncovers various roles of pol ζ that extend beyond translesion synthesis. For instance, pol ζ is also employed when the replisome operates sub-optimally or at difficult-to-replicate DNA sequences. Pol ζ also participates in repair by microhomology mediated break-induced replication. A rev3 deletion is tolerated in yeast but Rev3l disruption results in embryonic lethality in mice. Inactivation of mammalian Rev3l results in genomic instability and invokes cell death and senescence programs. Targeting of pol ζ function may be a useful strategy in cancer therapy, although chromosomal instability associated with pol ζ deficiency must be considered.

Kinetic Investigation of Translesion Synthesis across a 3-Nitrobenzanthrone-Derived DNA Lesion Catalyzed by Human DNA Polymerase Kappa.

3-Nitrobenzanthrone (3-NBA) is a byproduct of diesel exhaust and is highly present in industrial and populated areas. Inhalation of 3-NBA results in formation of N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dGC8-N-ABA), a bulky DNA lesion that is of concern due to its mutagenic and carcinogenic potential. If dGC8-N-ABA is not bypassed during genomic replication, the lesion can stall cellular DNA replication machinery, leading to senescence or apoptosis. We have previously used running start assays to demonstrate that human DNA polymerases eta (hPolη) and kappa (hPolκ) are able to catalyze translesion DNA synthesis (TLS) across a site-specifically placed dGC8-N-ABA in a DNA template. Consistently, gene knockdown of hPolη and hPolκ in HEK293T cells reduces the efficiency of TLS across dGC8-N-ABA by ∼25 and ∼30%, respectively. Here, we kinetically investigated why hPolκ paused when bypassing and extending from dGC8-N-ABA. Our kinetic data show that correct dCTP incorporation efficiency of hPolκ dropped by 116-fold when opposite dGC8-N-ABA relative to undamaged dG, leading to hPolκ pausing at the lesion site observed in the running start assays. The already low nucleotide incorporation fidelity of hPolκ was further decreased by 10-fold during lesion bypass, and thus, incorrect nucleotides, especially dATP, were incorporated opposite dGC8-N-ABA with comparable efficiencies as correct dCTP. With regard to the dGC8-N-ABA bypass product extension step, hPolκ incorporated correct dGTP onto the damaged DNA substrate with a 786-fold lower efficiency than onto the corresponding undamaged DNA substrate, which resulted in hPolκ pausing at the site in the running start assays. Furthermore, hPolκ extended the primer-terminal matched base pair dC:dGC8-N-ABA with a 100-1000-fold lower fidelity than it extended the undamaged dC:dG base pair. Together, our kinetic results strongly indicate that hPolκ was error-prone during TLS of dGC8-N-ABA.

PrimPol is required for the maintenance of efficient nuclear and mitochondrial DNA replication in human cells.

Eukaryotic Primase-Polymerase (PrimPol) is an enzyme that maintains efficient DNA duplication by repriming replication restart downstream of replicase stalling lesions and structures. To elucidate the cellular requirements for PrimPol in human cells, we generated PrimPol-deleted cell lines and show that it plays key roles in maintaining active replication in both the nucleus and mitochondrion, even in the absence of exogenous damage. Human cells lacking PrimPol exhibit delayed recovery after UV-C damage and increased mutation frequency, micronuclei and sister chromatin exchanges but are not sensitive to genotoxins. PrimPol is also required during mitochondrial replication, with PrimPol-deficient cells having increased mtDNA copy number but displaying a significant decrease in replication. Deletion of PrimPol in XPV cells, lacking functional polymerase Eta, causes an increase in DNA damage sensitivity and pronounced fork stalling after UV-C treatment. We show that, unlike canonical TLS polymerases, PrimPol is important for allowing active replication to proceed, even in the absence of exogenous damage, thus preventing the accumulation of excessive fork stalling and genetic mutations. Together, these findings highlight the importance of PrimPol for maintaining efficient DNA replication in unperturbed cells and its complementary roles, with Pol Eta, in damage tolerance in human cells.

DNA polymerase θ (POLQ) is important for repair of DNA double-strand breaks caused by fork collapse.

DNA polymerase θ (POLQ) plays an important role in alternative nonhomologous end joining or microhomology-mediated end joining (alt-NHEJ/MMEJ). Here, we show that POLQ is not only required for MMEJ to repair DNA double-strand breaks (DSBs) generated by endonucleases such as I-SceI or Cas9, but is also needed for repair of DSBs derived from DNA nicks generated by Cas9 nickase. Consistently, we found that POLQ deficiency leads to sensitivity to topoisomerase inhibitors that cause DNA single-strand break (SSB) accumulation at replication forks and to ATR inhibitors that induce replication fork collapse. These studies support the function of POLQ in coping with replication stress and repairing DSBs upon fork collapse. POLQ overexpression is present in many cancer types and is associated with poor prognosis, including breast cancer regardless of BRCA1 status. We provide proof-of-concept evidence to support a novel cancer treatment strategy that combines POLQ inhibition with administration of topoisomerase or ATR inhibitors, which induces replication stress and fork collapse. Given the prevalence of POLQ overexpression in tumors, such strategy may have a significant impact on developing targeted cancer treatment.

MtDNA mutagenesis impairs elimination of mitochondria during erythroid maturation leading to enhanced erythrocyte destruction.

Haematopoietic progenitor cells show special sensitivity to mitochondrial DNA (mtDNA) mutagenesis, which suggests that increased mtDNA mutagenesis could underlie anemias. Here we show that elevated mtDNA mutagenesis in mice with a proof-reading deficient mtDNA polymerase (PolG) leads to incomplete mitochondrial clearance, with asynchronized iron loading in erythroid precursors, and increased total and free cellular iron content. The resulting Fenton chemistry leads to oxidative damage and premature destruction of erythrocytes by splenic macrophages. Our data indicate that mitochondria actively contribute to their own elimination in reticulocytes and modulate iron loading. Asynchrony of this sequence of events causes severe mitochondrial anaemia by depleting the organism of red blood cells and the bone marrow of iron. Our findings account for the anaemia development in a progeroid mouse model and may have direct relevance to the anemias associated with human mitochondrial disease and ageing.

Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination.

The alternative non-homologous end-joining (NHEJ) machinery facilitates several genomic rearrangements, some of which can lead to cellular transformation. This error-prone repair pathway is triggered upon telomere de-protection to promote the formation of deleterious chromosome end-to-end fusions. Using next-generation sequencing technology, here we show that repair by alternative NHEJ yields non-TTAGGG nucleotide insertions at fusion breakpoints of dysfunctional telomeres. Investigating the enzymatic activity responsible for the random insertions enabled us to identify polymerase theta (Polθ; encoded by Polq in mice) as a crucial alternative NHEJ factor in mammalian cells. Polq inhibition suppresses alternative NHEJ at dysfunctional telomeres, and hinders chromosomal translocations at non-telomeric loci. In addition, we found that loss of Polq in mice results in increased rates of homology-directed repair, evident by recombination of dysfunctional telomeres and accumulation of RAD51 at double-stranded breaks. Lastly, we show that depletion of Polθ has a synergistic effect on cell survival in the absence of BRCA genes, suggesting that the inhibition of this mutagenic polymerase represents a valid therapeutic avenue for tumours carrying mutations in homology-directed repair genes.

Homologous-recombination-deficient tumours are dependent on Polθ-mediated repair.

Large-scale genomic studies have shown that half of epithelial ovarian cancers (EOCs) have alterations in genes regulating homologous recombination (HR) repair. Loss of HR accounts for the genomic instability of EOCs and for their cellular hyper-dependence on alternative poly-ADP ribose polymerase (PARP)-mediated DNA repair mechanisms. Previous studies have implicated the DNA polymerase θ (Polθ also known as POLQ, encoded by POLQ) in a pathway required for the repair of DNA double-strand breaks, referred to as the error-prone microhomology-mediated end-joining (MMEJ) pathway. Whether Polθ interacts with canonical DNA repair pathways to prevent genomic instability remains unknown. Here we report an inverse correlation between HR activity and Polθ expression in EOCs. Knockdown of Polθ in HR-proficient cells upregulates HR activity and RAD51 nucleofilament assembly, while knockdown of Polθ in HR-deficient EOCs enhances cell death. Consistent with these results, genetic inactivation of an HR gene (Fancd2) and Polq in mice results in embryonic lethality. Moreover, Polθ contains RAD51 binding motifs and it blocks RAD51-mediated recombination. Our results reveal a synthetic lethal relationship between the HR pathway and Polθ-mediated repair in EOCs, and identify Polθ as a novel druggable target for cancer therapy.

Berberine induces double-strand DNA breaks in Rev3 deficient cells.

Berberine is a natural isoquinoline alkaloid, the majority of which is extracted from Huang Lian and other medicinal herbs. Numerous studies have revealed that berberine exhibits anticancer activity, however the mechanisms underlying this effect remain elusive. To examine these mechanisms, we analyzed the effects of berberine on a panel of DNA repair deficient chicken B lymphocyte (DT40) clones. Our results revealed that DT40 cells deficient in Rev3 (Rev3-/-), a translesion DNA synthesis (TLS) gene, were hypersensitive to berberine. Following berberine treatment, cell cycle analysis identified that G2/M arrest was increased in Rev3-/- cells. Furthermore, compared with wild-type cells (WT), berberine also induced a significant increase in double-strand breaks (DSBs) in Rev3-/- cells, as revealed by chromosomal aberration (CA) analysis. These results suggest that berberine is able to induce DNA damage, and that the Rev3 associated DNA repair pathway participates in the processes that aid its repair.

TRIP/NOPO E3 ubiquitin ligase promotes ubiquitylation of DNA polymerase η.

We previously identified a Drosophila maternal effect-lethal mutant named 'no poles' (nopo). Embryos from nopo females undergo mitotic arrest with barrel-shaped, acentrosomal spindles during the rapid cycles of syncytial embryogenesis because of activation of a Chk2-mediated DNA checkpoint. NOPO is the Drosophila homolog of human TNF receptor associated factor (TRAF)-interacting protein (TRIP), which has been implicated in TNF signaling. NOPO and TRIP contain RING domains closely resembling those of known E3 ubiquitin ligases. We herein sought to elucidate the mechanism by which TRIP/NOPO promotes genomic stability by performing a yeast two-hybrid screen to identify potential substrates/interactors. We identified members of the Y-family of DNA polymerases that facilitate replicative bypass of damaged DNA (translesion synthesis) as TRIP interactors. We show that TRIP and NOPO co-immunoprecipitate with human and Drosophila Polη, respectively, from cultured cells. We generated a null mutation in Drosophila Polη (dPolη) and found that dPolη-derived embryos have increased sensitivity to ultraviolet irradiation and exhibit nopo-like mitotic spindle defects. dPolη and nopo interact genetically in that overexpression of dPolη in hypomorphic nopo-derived embryos suppresses nopo phenotypes. We observed enhanced ubiquitylation of Polη by TRIP and NOPO E3 ligases in human cells and Drosophila embryos, respectively, and show that TRIP promotes hPolη localization to nuclear foci in human cells. We present a model in which TRIP/NOPO ubiquitylates Polη to positively regulate its activity in translesion synthesis.

The error-prone DNA polymerase ι provides quantitative resistance to lung tumorigenesis and mutagenesis in mice.

Opposite undamaged nucleotide T, DNA polymerase ι (Polι) preferentially incorporates G rather than A, violating the Watson-Crick rule. Although the actual biological role of Polι remains enigmatic, we have identified its coding gene as a candidate for pulmonary adenoma resistance 2 (Par2), a mouse quantitative trait locus modulating chemically induced lung tumor susceptibility. Notably, the most tumor-sensitive Par2 allele possessed by the 129X1/SvJ mouse is associated with a loss-of-function mutation in Polι. To determine whether the nonfunctional Polι is responsible for the 129X1/SvJ-specific Par2 phenotype, we knocked out Polι in a C57BL/6J mouse carrying a less tumor-sensitive Par2 allele. Disruption of the C57BL/6J Polι conferred 129X1/SvJ-like sensitivity on the C57BL/6J Par2 locus and increased the in vivo mutation frequency in the lung, providing definitive proof that Polι causes the Par2 effect and inhibits tumorigenesis and mutagenesis, despite its extreme replication infidelity.

DNA polymerase η modulates replication fork progression and DNA damage responses in platinum-treated human cells.

Human cells lacking DNA polymerase η (polη) are sensitive to platinum-based cancer chemotherapeutic agents. Using DNA combing to directly investigate the role of polη in bypass of platinum-induced DNA lesions in vivo, we demonstrate that nascent DNA strands are up to 39% shorter in human cells lacking polη than in cells expressing polη. This provides the first direct evidence that polη modulates replication fork progression in vivo following cisplatin and carboplatin treatment. Severe replication inhibition in individual platinum-treated polη-deficient cells correlates with enhanced phosphorylation of the RPA2 subunit of replication protein A on serines 4 and 8, as determined using EdU labelling and immunofluorescence, consistent with formation of DNA strand breaks at arrested forks in the absence of polη. Polη-mediated bypass of platinum-induced DNA lesions may therefore represent one mechanism by which cancer cells can tolerate platinum-based chemotherapy.

Polymerase gamma deficiency (POLG): clinical course in a child with a two stage evolution from infantile myocerebrohepatopathy spectrum to an Alpers syndrome and neuropathological findings of Leigh's encephalopathy.

AIMS: Description of the clinical course in a child compound heterozygous for POLG1 mutations, neuropathology findings and results of dietary treatment based on fasting avoidance and long chain triglycerides (LCT) restriction. RESULTS: At 3(1/2) months of age the patient presented with severe hypoglycemia, hyperlactatemia, moderate ketosis and hepatic failure. Fasting hypoglycemia occurred 8 h after meals. The hypoglycemia did not respond to glucagon. She was supplemented with IV glucose and/or frequent feedings, but developed liver insufficiency which was reversed by long-chain triglyceride (LCT) restriction. Alpha-foeto-protein (AFP) levels were elevated and returned to low values after dietary treatment. Liver biopsy displayed cirrhosis, bile ductular proliferation, steatosis, isolated complex IV defect in part of the liver mitochondria, and mitochondrial DNA depletion (27% of control values). Two heterozygous mutations (p. [Ala467Thr] + p. [Gly848Ser]) were found in the POLG1 gene. At 3 years of age she progressively developed refractory mixed type seizures including a focal component and psychomotor regression which fulfilled the criteria of Alpers syndrome (AS) although the initial presentation was compatible with infantile myocerebrohepatopathy spectrum (MCHS). She died at 5 years of age of respiratory insufficiency. Neuropathologic investigation revealed lesions in the right striatal area and the inferior colliculi typical for Leigh's encephalopathy. CONCLUSION: The present patient showed an evolution from infantile MCHS to AS, and dietary treatment seemed to slow the progression of liver failure. In spite of the late clinical features of AS, it extends the neuropathological spectrum of AS and polymerase gamma deficiency (POLG) to Leigh syndrome lesions.

Stalled Polη at its cognate substrate initiates an alternative translesion synthesis pathway via interaction with REV1.

DNA polymerase η (Polη), whose gene mutation is responsible for the inherited disorder xeroderma pigmentosum variant (XP-V), carries out accurate and efficient translesion synthesis (TLS) across cyclobutane pyrimidine dimer (CPD). As Polη interacts with REV1, and REV1 interacts with other TLS polymerases including Polι, Polκ and Polζ, Polη may play a role in recruitment of these TLS polymerases at lesion site. But it is unclear whether UV sensitivity of XP-V patients is caused not only by defect of Polη activity but also by dysfunction of network between Polη and other TLS polymerases. Here, we examined whether the TLS polymerase network via Polη is important for replicative bypass of CPDs and DNA damage tolerance induced by UV in mouse cells. We observed that UV sensitivity of Polη-deficient mouse cells was moderately rescued by the expression of a catalytically inactive Polη. Moreover, this recovery of cellular UV sensitivity was mediated by the interaction between Polη and REV1. However, expression of the inactive mutant Polη was not able to suppress the incidence of UV-induced mutation observed in Polη-deficient cells. We propose the model that REV1 and Polκ are involved in DNA damage tolerance via Polη-REV1 interaction when Polη fails to bypass its cognate substrates.

Suppression of Rev3, the catalytic subunit of Pol{zeta}, sensitizes drug-resistant lung tumors to chemotherapy.

Platinum-based chemotherapeutic drugs are front-line therapies for the treatment of non-small cell lung cancer. However, intrinsic drug resistance limits the clinical efficacy of these agents. Recent evidence suggests that loss of the translesion polymerase, Polζ, can sensitize tumor cell lines to cisplatin, although the relevance of these findings to the treatment of chemoresistant tumors in vivo has remained unclear. Here, we describe a tumor transplantation approach that enables the rapid introduction of defined genetic lesions into a preclinical model of lung adenocarcinoma. Using this approach, we examined the effect of impaired translesion DNA synthesis on cisplatin response in aggressive late-stage lung cancers. In the presence of reduced levels of Rev3, an essential component of Polζ, tumors exhibited pronounced sensitivity to cisplatin, leading to a significant extension in overall survival of treated recipient mice. Additionally, treated Rev3-deficient cells exhibited reduced cisplatin-induced mutation, a process that has been implicated in the induction of secondary malignancies following chemotherapy. Taken together, our data illustrate the potential of Rev3 inhibition as an adjuvant therapy for the treatment of chemoresistant malignancies, and highlight the utility of rapid transplantation methodologies for evaluating mechanisms of chemotherapeutic resistance in preclinical settings.

Lack of DNA polymerase µ affects the kinetics of DNA double-strand break repair and impacts on cellular senescence.

The specialised DNA polymerase µ (pol µ) affects a sub-class of immunoglobulin genes rearrangements and haematopoietic development in vivo. These effects appear linked to double-strand breaks (DSBs) repair, but it is still unclear how and to what extent pol µ intervenes in this process. Using high-resolution quantitative imaging of DNA damage in irradiated wild-type and pol µâ»(/)⁻ mouse embryonic fibroblasts (MEFs) we show that lack of pol µ results in delayed DSB repair kinetics and in persistent DNA damage. DNA damage triggers cellular senescence, and this response is thought to suppress cancer. Independent investigations either report or not a proliferative decline for MEFs lacking pol µ. Here we show pronounced senescence in pol µâ»(/)⁻ MEFs, associated with high levels of the tumor-suppressor p16(INK4A) and the DNA damage response kinase CHK2. Importantly, cellular senescence is induced by culture stress and exacerbated by low doses of irradiation in pol µâ»(/)⁻ MEFs. We also found that low doses of irradiation provoke delayed immortalisation in MEFs lacking pol µ. Pol µâ»(/)⁻ MEFs thus exhibit a robust anti-proliferative defence in response to irreparable DNA damage. These findings indicate that sub-optimal DSB repair, due to the absence of an auxiliary DNA damage repair factor, can impact on cell fitness and thereby on cell fate.

A small interfering RNA screen of genes involved in DNA repair identifies tumor-specific radiosensitization by POLQ knockdown.

The effectiveness of radiotherapy treatment could be significantly improved if tumor cells could be rendered more sensitive to ionizing radiation (IR) without altering the sensitivity of normal tissues. However, many of the key therapeutically exploitable mechanisms that determine intrinsic tumor radiosensitivity are largely unknown. We have conducted a small interfering RNA (siRNA) screen of 200 genes involved in DNA damage repair aimed at identifying genes whose knockdown increased tumor radiosensitivity. Parallel siRNA screens were conducted in irradiated and unirradiated tumor cells (SQ20B) and irradiated normal tissue cells (MRC5). Using gammaH2AX foci at 24 hours after IR, we identified several genes, such as BRCA2, Lig IV, and XRCC5, whose knockdown is known to cause increased cell radiosensitivity, thereby validating the primary screening end point. In addition, we identified POLQ (DNA polymerase ) as a potential tumor-specific target. Subsequent investigations showed that POLQ knockdown resulted in radiosensitization of a panel of tumor cell lines from different primary sites while having little or no effect on normal tissue cell lines. These findings raise the possibility that POLQ inhibition might be used clinically to cause tumor-specific radiosensitization.

Loss of DNA polymerase zeta enhances spontaneous tumorigenesis.

Mammalian genomes encode at least 15 distinct DNA polymerases, functioning as specialists in DNA replication, DNA repair, recombination, or bypass of DNA damage. Although the DNA polymerase zeta (polzeta) catalytic subunit REV3L is important in defense against genotoxins, little is known of its biological function. This is because REV3L is essential during embryogenesis, unlike other translesion DNA polymerases. Outstanding questions include whether any adult cells are viable in the absence of polzeta and whether polzeta status influences tumorigenesis. REV3L-deficient cells have properties that could influence the development of neoplasia in opposing ways: markedly reduced damage-induced point mutagenesis and extensive chromosome instability. To answer these questions, Rev3L was conditionally deleted from tissues of adult mice using MMTV-Cre. Loss of REV3L was tolerated in epithelial tissues but not in the hematopoietic lineage. Thymic lymphomas in Tp53(-/-) Rev3L conditional mice occurred with decreased latency and higher incidence. The lymphomas were populated predominantly by Rev3L-null T cells, showing that loss of Rev3L can promote tumorigenesis. Remarkably, the tumors were frequently oligoclonal, consistent with accelerated genetic changes in the absence of Rev3L. Mammary tumors could also arise from Rev3L-deleted cells in both Tp53(+/+) and Tp53(+/-) backgrounds. Mammary tumors in Tp53(+/-) mice deleting Rev3L formed months earlier than mammary tumors in Tp53(+/-) control mice. Prominent preneoplastic changes in glandular tissue adjacent to these tumors occurred only in mice deleting Rev3L and were associated with increased tumor multiplicity. Polzeta is the only specialized DNA polymerase yet identified that inhibits spontaneous tumor development.