Alteration in the DNA replication, repair or recombination processes is a highly relevant mechanism of genomic instability. Despite genomic aberrations manifested in hematologic malignancies, such a defect as a source of biomarkers has been underexplored. Here, we investigated the prognostic value of expression of 82 genes involved in DNA replication-repair-recombination in a series of 99 patients with chronic lymphocytic leukemia without detectable 17p deletion or TP53 mutation. We found that expression of the POLN gene, encoding the specialized DNA polymerase ν (Pol ν) correlates with time to relapse after first-line therapy with fludarabine. Moreover, we found that POLN was the only gene up-regulated in primary patients' lymphocytes when exposed in vitro to proliferative and pro-survival stimuli. By using two cell lines that were sequentially established from the same patient during the course of the disease and Pol ν knockout mouse embryonic fibroblasts, we reveal that high relative POLN expression is important for DNA synthesis and cell survival upon fludarabine treatment. These findings suggest that Pol ν could influence therapeutic resistance in chronic lymphocytic leukemia. (Patients' samples were obtained from the CLL 2007 FMP clinical trial registered at: clinicaltrials.gov identifer: 00564512).

Adipose-derived stem cells (ADSCs) have led to growing interest in cell-based therapy because they can be easily harvested from an abundant tissue. ADSCs must be expanded in vitro before transplantation. This essential step causes concerns about the safety of adult stem cells in terms of potential transformation. Tumorigenesis is driven in its earliest step by DNA replication stress, which is characterized by the accumulation of stalled DNA replication forks and activation of the DNA damage response. Thus, to evaluate the safety of ADSCs during ex vivo expansion, we monitored DNA replication under atmospheric (21%) or physiologic (1%) oxygen concentration. Here, by combining immunofluorescence and DNA combing, we show that ADSCs cultured under 21% oxygen accumulate endogenous oxidative DNA lesions, which interfere with DNA replication by increasing fork stalling events, thereby leading to incomplete DNA replication and fork collapse. Moreover, we found by RNA sequencing (RNA-seq) that culture of ADSCs under atmospheric oxygen concentration leads to misexpression of cell cycle and DNA replication genes, which could contribute to DNA replication stress. Finally, analysis of acquired small nucleotide polymorphism shows that expansion of ADSCs under 21% oxygen induces a mutational bias toward deleterious transversions. Overall, our results suggest that expanding ADSCs at a low oxygen concentration could reduce the risk for DNA replication stress-associated transformation, as occurs in neoplastic tissues.

Oncogenic transcription factors are commonly activated in acute leukemias and subvert normal gene expression networks to reprogram hematopoietic progenitors into preleukemic stem cells, as exemplified by LIM-only 2 (LMO2) in T-cell acute lymphoblastic leukemia (T-ALL). Whether or not these oncoproteins interfere with other DNA-dependent processes is largely unexplored. Here, we show that LMO2 is recruited to DNA replication origins by interaction with three essential replication enzymes: DNA polymerase delta (POLD1), DNA primase (PRIM1), and minichromosome 6 (MCM6). Furthermore, tethering LMO2 to synthetic DNA sequences is sufficient to transform these sequences into origins of replication. We next addressed the importance of LMO2 in erythroid and thymocyte development, two lineages in which cell cycle and differentiation are tightly coordinated. Lowering LMO2 levels in erythroid progenitors delays G1-S progression and arrests erythropoietin-dependent cell growth while favoring terminal differentiation. Conversely, ectopic expression in thymocytes induces DNA replication and drives these cells into cell cycle, causing differentiation blockade. Our results define a novel role for LMO2 in directly promoting DNA synthesis and G1-S progression.

The nucleoside analog cytarabine, an inhibitor of DNA replication fork progression that results in DNA damage, is currently used in the treatment of acute myeloid leukemia (AML). We explored the prognostic value of the expression of 72 genes involved in various aspects of DNA replication in a set of 198 AML patients treated by cytarabine-based chemotherapy. We unveiled that high expression of the DNA replication checkpoint gene CHEK1 is a prognostic marker associated with shorter overall, event-free, and relapse-free survivals and determined that the expression of CHEK1 can predict more frequent and earlier postremission relapse. CHEK1 encodes checkpoint kinase 1 (CHK1), which is activated by the kinase ATR when DNA replication is impaired by DNA damage. High abundance of CHK1 in AML patient cells correlated with higher clonogenic ability and more efficient DNA replication fork progression upon cytarabine treatment. Exposing the patient cells with the high abundance of CHK1 to SCH900776, an inhibitor of the kinase activity of CHK1, reduced clonogenic ability and progression of DNA replication in the presence of cytarabine. These results indicated that some AML cells rely on an efficient CHK1-mediated replication stress response for viability and that therapeutic strategies that inhibit CHK1 could extend current cytarabine-based treatments and overcome drug resistance. Furthermore, monitoring CHEK1 expression could be used both as a predictor of outcome and as a marker to select AML patients for CHK1 inhibitor treatments.

DNA polymerase theta (Polθ) is a specialized A-family DNA polymerase that functions in processes such as translesion synthesis (TLS), DNA double-strand break repair and DNA replication timing. Overexpression of POLQ, the gene encoding Polθ, is a prognostic marker for an adverse outcome in a wide range of human cancers. While increased Polθ dosage was recently suggested to promote survival of homologous recombination (HR)-deficient cancer cells, it remains unclear whether POLQ overexpression could be also beneficial to HR-proficient cancer cells. By performing a short interfering (si)RNA screen in which genes encoding druggable proteins were knocked down in Polθ-overexpressing cells as a means to uncover genetic vulnerabilities associated with POLQ overexpression, we could not identify genes that were essential for viability in Polθ-overexpressing cells in normal growth conditions. We also showed that, upon external DNA replication stress, Polθ expression promotes cell survival and limits genetic instability. Finally, we report that POLQ expression correlates with the expression of a set of HR genes in breast, lung and colorectal cancers. Collectively, our data suggest that Polθ upregulation, besides its importance for survival of HR-deficient cancer cells, may be crucial also for HR-proficient cells to better tolerate DNA replication stress, as part of a global gene deregulation response, including HR genes.

The levels of the cyclin-dependent kinase (CDK) inhibitor p21 are low in S phase and insufficient to inhibit CDKs. We show here that endogenous p21, instead of being residual, it is functional and necessary to preserve the genomic stability of unstressed cells. p21depletion slows down nascent DNA elongation, triggers permanent replication defects and promotes the instability of hard-to-replicate genomic regions, namely common fragile sites (CFS). The p21's PCNA interacting region (PIR), and not its CDK binding domain, is needed to prevent the replication defects and the genomic instability caused by p21 depletion. The alternative polymerase kappa is accountable for such defects as they were not observed after simultaneous depletion of both p21 and polymerase kappa. Hence, in CDK-independent manner, endogenous p21 prevents a type of genomic instability which is not triggered by endogenous DNA lesions but by a dysregulation in the DNA polymerase choice during genomic DNA synthesis.

One of the hallmarks of cancer is the occurrence of high levels of chromosomal rearrangements as a result of inaccurate repair of double-strand breaks (DSB). Germline mutations in BRCA and RAD51 genes, involved in DSB repair, are strongly associated with hereditary breast cancer. Pol θ, a translesional DNA polymerase specialized in the replication of damaged DNA, has been also shown to contribute to DNA synthesis associated to DSB repair. It is noteworthy that POLQ is highly expressed in breast tumors and this expression is able to predict patient outcome. The objective of this study was to analyze genetic variants related to POLQ as new population biomarkers of risk in hereditary (HBC) and sporadic (SBC) breast cancer.

It remains presently unclear whether disease progression in colorectal carcinoma (CRC), from early, to invasive and metastatic forms, is associated to a gradual increase in genetic instability and to a scheme of sequentially occurring Copy Number Alterations (CNAs).

BACKGROUND:Colorectal and breast cancers are among the most common cancers worldwide. They result from a conjugated deficiency of gene maintenance and cell cycle control.OBJECTIVE:We investigate the expression of the microtubule-associated protein MAP9/ASAP and its two partners AURKA and PLK1 in colorectal tumors as well as in ductal breast cancers.MATERIALS AND METHODS:26 colorectal cancer samples and adjacent normal tissues and 77 ductal breast cancer samples from grade I to grade III were collected. Real-time quantitative PCR was used to analyse the expression of MAP9, AURKA, and PLK1. Results. Expression of MAP9 is downregulated in colorectal cancer compared to normal tissues (P > 10(-3)), whereas those of AURKA and PLK1 are upregulated (P > 10(-4)). In ductal breast cancer, we found a grade-dependent increase of AURKA expression (P > 10(-3)), while the variations of expression of MAP9 and PLK1 are not significant (P > 0.2).CONCLUSIONS:MAP9 downregulation is associated with colorectal malignancy and could be used as a disease marker and a new drug target, while AURKA and PLK1 are upregulated. In ductal breast cancer, AURKA overexpression is strongly associated with the tumor grade and is therefore of prognostic value for the progression of the disease.

Although DNA polymerase θ (Pol θ) is known to carry out translesion synthesis and has been implicated in DNA repair, its physiological function under normal growth conditions remains unclear. Here we present evidence that Pol θ plays a role in determining the timing of replication in human cells. We find that Pol θ binds to chromatin during early G1, interacts with the Orc2 and Orc4 components of the Origin recognition complex and that the association of Mcm proteins with chromatin is enhanced in G1 when Pol θ is downregulated. Pol θ-depleted cells exhibit a normal density of activated origins in S phase, but early-to-late and late-to-early shifts are observed at a number of replication domains. Pol θ overexpression, on the other hand, causes delayed replication. Our results therefore suggest that Pol θ functions during the earliest steps of DNA replication and influences the timing of replication initiation.

Space debris mitigation is one objective of the French Space Operations Act (FSOA), in line with Inter-Agency Space Debris Coordination Committee (IADC) recommendations, through the removal of non-operational objects from populated regions. At the end of their mission, space objects are to be placed on orbits that will minimize future hazards to space objects orbiting in the same region. The FSOA, which came into force in 2010, ensures that technical risks associated with space activities are properly mitigated. The Act confers CNES a central support role in providing technical expertise to government on regulations dealing with space operations. In order to address the compliance of disposal orbits with the law technical requirements, CNES draws up Good Practices as well as a dedicated tool, Semi-analytic Tool for End of Life Analysis (STELA).The verification of the criteria of the French Space Operations Act requires long term orbit propagation to evaluate the evolution of the orbital elements over long time scales (up to more than 100 years). The Good Practices define the minimum dynamical model required to compute the orbital evolution with sufficient accuracy, and detail key computation hypotheses such as drag and reflecting areas, drag coefficient, reflectivity coefficient, solar activity, atmospheric density model and so on. They also recommend a methodology adapted to each orbit type (LEO, GEO, GTO) to assess the criteria of the French Space Operations Act. The most recent works have concerned GTO, for which some couplings occur between dynamic perturbations. A small change in the initial conditions or in the estimation of the drag effect will significantly change the entrance conditions in resonance areas and thus the orbital evolution. To cope with this difficulty, a statistical method has been developed.This paper gives an overview of the Good Practices for orbit propagation in LEO, GEO and GTO as well as a brief description of the STELA tool. It explains the specificities of GTO and the need for a statistical approach, through a Monte-Carlo campaign of orbital propagations. Then, it raises the question of the statistical convergence and proposes a methodology to estimate a confidence interval for the results. Finally, special cases consisting of typical GTO are treated. (C) 2013 IAA. Published by Elsevier Ltd. All rights reserved.

Formation of primed single‐stranded DNA at stalled replication forks triggers activation of the replication checkpoint signalling cascade resulting in the ATR‐mediated phosphorylation of the Chk1 protein kinase, thus preventing genomic instability. By using siRNA‐mediated depletion in human cells and immunodepletion and reconstitution experiments in Xenopus egg extracts, we report that the Y‐family translesion (TLS) DNA polymerase kappa (Pol κ) contributes to the replication checkpoint response and is required for recovery after replication stress. We found that Pol κ is implicated in the synthesis of short DNA intermediates at stalled forks, facilitating the recruitment of the 9‐1‐1 checkpoint clamp. Furthermore, we show that Pol κ interacts with the Rad9 subunit of the 9‐1‐1 complex. Finally, we show that this novel checkpoint function of Pol κ is required for the maintenance of genomic stability and cell proliferation in unstressed human cells. A vertebrate translesion synthesis DNA polymerase broadly contributes to checkpoint‐activating primer synthesis at stalled replication forks, a role previously ascribed only to replicative polymerases.

In addition to the canonical right-handed double helix, DNA molecule can adopt several other non-B DNA structures. Readily formed in the genome at specific DNA repetitive sequences, these secondary conformations present a distinctive challenge for progression of DNA replication forks. Impeding normal DNA synthesis, cruciforms, hairpins, H DNA, Z DNA and G4 DNA considerably impact the genome stability and in some instances play a causal role in disease development. Along with previously discovered dedicated DNA helicases, the specialized DNA polymerases emerge as major actors performing DNA synthesis through these distorted impediments. In their new role, they are facilitating DNA synthesis on replication stalling sites formed by non-B DNA structures and thereby helping the completion of DNA replication, a process otherwise crucial for preserving genome integrity and concluding normal cell division. This review summarizes the evidence gathered describing the function of specialized DNA polymerases in replicating DNA through non-B DNA structures.

line 8: 'stalled forks' Page 4, line 2: extra '(' Page 16, line 4: extra '-' after pol kappa. Appeal 10 January 2013 We thank you very much for having considered our manuscript by Bétous et al., “DNA polymerase Kappa is implicated in continued DNA synthesis at stalled replication forks and is required for checkpoint activation” (manuscript EMBOJ-2012-83985) for publication in EMBO Journal. We have greatly appreciated your effort for having coordinated the reviewers’ reports that we have now thoroughly analyzed. Although reviewer #1 opinion (which is most arguable throughout most of the criticisms raised, see below) is against publication, reviewers #2 and #3 find the work of interest and of importance in the field and recommend publication after taking into consideration the points they have raised, and that we are very much willing to satisfy. Reviewer #2 has only a few minor points to be clarified, some of them are just clarifications of experiments performed in xenopus egg extracts as it appears that he/she ‘s not an expert with this system. Reviewer #3 requires a clarification of previous data reported by Bi et al., using polk-/MEFs showing activation of the checkpoint after exposure to a DNA damaging agent (BPDE) in these cells, and we would be very happy to address and explain the apparent discrepancy and include new data with the Pol κ MEFs that confirm that Pol k is required The EMBO Journal Peer Review Process File EMBO-2012-83985 © European Molecular Biology Organization 5 for the activation of the replication checkpoint after HU, as we found in human cells and Xenopus extracts. As for reviewer #1, we do not understand the meaning of most of the points raised (point 2, 3, 5, 6, 9a and 9b), and the other remaining points are very arguable and not justified, some of them can be satisfied (see comments below point by point at the end of our letter). The major point the reviewer #1 raises is the relationship between Pol κ and the replicative Pol δ regarding the generation of the short fragments at stalled replication forks after aphidicolin, important for the checkpoint activation. He/She pointed out (see point 7 of reviewer#1) the apparent discrepancy with the work of Van et al., (JCB. 2010) which showed the requirement for Pol δ in the synthesis of these small replication intermediates. In particular the reviewer argues that “Van et al. showed that the generation of short fragments is almost completely dependent on Pol δ, while the authors show that it is almost completely dependent on Pol κ”. This argument is not justified, since depletion of neither Pol δ nor Pol κ results in a complete disappearance of the short fragments (see Fig 5C-lane ΔPol δ/90 min in Van et al. and Fig 3C from our manuscript, lane 90 min), nor in a total decrease of P-Chk1 (see Fig 6B from Van et al and Fig 1A-B from our manuscript). Our observations show that, besides Pol δ, another polymerase is implicated at replication forks stalled after aphidicolin treatment, and underline the complexity of the system. According to us, there is no discrepancy between the two works. Moreover, as also stated in our manuscript, it has to be remembered that this finding is in agreement with the observation that, in contrast to the three replicative DNA polymerases Pol δ, Pol ε, and Pol α, Pol κ is not inhibited by aphidicolin, and therefore is one DNA polymerase active at arrested forks. We cannot exclude that both polymerases act in a mutually dependent and coordinative manner. We do not think that repeating the experiment described in Van et al., consisting in removing Pol δ from egg extracts, as asked by the reviewer #1, is justified and will be very informative. By the way, in Supplementary Figure 2A and Figure 3E of our manuscript we have shown that Pol k binds to chromatin in S-phase only in the presence of aphidicolin, which demonstrates that loading of Pol k occurs after that of Pol δ since Pol δ is already bound to chromatin in S-phase. Moreover, it is known that loading of the 9-1-1 checkpoint clamp requires formation of replication intermediates long enough to be recognized by the RFC-Rad17 complex that loads the 9-1-1 clamp (Ellison and Stillman 2003. Plos Biology). These intermediates are made by the concerted and sequential action of Pol α (RNA primers) and Pol δ and it is very unlikely that Pol κ can do this job independently of Pol δ since Pol κ is not processive and can synthesize only very few nucleotides. We believe that Pol κ functions on the replication intermediates made by Pol δ to restrain excessive DNA synthesis and to stabilize the short replication intermediates that are bound by the 9-1-1 complex. It is likely that in the absence of Pol κ these intermediates are not stable for reasons that we still do not understand and this model will require more work to be proven, and this is not in the scope of the paper. We can further clarify this point in the discussion. In conclusion we do not really understand the rational behind the reviewer #1’s request. These additional data will be anyway redundant with those provided by Van et al., and will certainly diminish the originality of the paper. The aim of our work has been to demonstrate a novel role for a translesion DNA polymerase (Pol κ) in checkpoint activation, which is a completely novel concept in the field, and not to study the epistasis of this polymerase with respect to the replicative DNA polymerases. Nevertheless, we can propose a couple of experiments that would give further insights into the relationships between Pol δ and Pol κ. The first one consists of using a high concentration of aphidicolin in xenopus egg extracts. In these conditions only Pol α and not Pol δ, neither PCNA, nor the 9-1-1 load onto chromatin at arrested forks, as previously shown by some of us and other labs (Michael et al., 2000 Science; Maiorano et al., Cell 2005; Byun et al 2005, Genes & Dev., Zembutsu and Waga, 2006 Nucl. Acids Res.). We can determine whether in these conditions Pol κ binds or not to chromatin. The second set of experiments would be to determine whether there's a physical interaction between Pol κ and Pol δ by immunoprecipitation and whether removal of Pol κ also removes Pol δ. In conclusion we are very much willing to provide more evidence required to convince the reviewer#1 that Pol κ is indeed an important element of the replication checkpoint and we are therefore writing to ask whether after taking into consideration all our arguments, you will be willing to give us the possibility to resubmit a revised version of our manuscript for publication in The EMBO Journal Peer Review Process File EMBO-2012-83985 © European Molecular Biology Organization 6 EMBO Journal and/or to contact a fourth reviewer who would be an expert of the Xenopus in vitro system (such as Dr. Karlen Cimprich, Stanford University. USA, for instance). Point by point answer to referee 1 Point 1. The experiments shown throughout the paper are representative examples of independent observations made several times and by different researchers. The level of proteins involved in the checkpoint has been checked after Pol κ si-RNA and can be showed in a revised version. Point 2. We do not understand at all this point. Even if the concentration of the primase Pol α would be higher, we removed only Pol κ and we demonstrate in Figure 2D that Pol κ is indeed not required for replication, which rules out the unlikely possibility that Pol κ and Pol α interact. Nevertheless we can investigate this point by immunoprecipitation. Point 3. Obviously, we have this data as previously shown by us and other lab (Recolin et al., 2012; Nucl. Acids Res.; Byun et al., 2005; Genes & Dev.; Lupardus et al., 2000 Genes & Dev.) and we can add this data to satisfy the referee. Point 4. We can add Chk1 as a loading control. Point 5. We do not understand this point either. Figure 2D clearly illustrates that the scale between the two graphs is identical. This experiment also shows that removal of Pol κ does not affect the rate of DNA synthesis and demonstrates that indeed Pol κ is not required for chromosomal DNA synthesis in the absence of aphidicolin, and this is also true in mammalian cells. We do reproducibly observe slightly more DNA synthesis in Pol κ-depleted extracts, which is very likely due to failure to activate the intra S phase checkpoint that restrains activation of late clusters of replication origins (Shechter et al., 2004. Nat. Cell Biol.). This is entirely consistent with the observation that caffein, an inhibitor of ATM/ATR, has no effect on DNA synthesis after removal of Pol κ (Figure 2D). Point 6. Figure 2D shows a representative replication assay performed with either mockor Pol κdepleted extracts, and also shows that indeed removal of Pol κ at the 90 minutes time point does not decreases the total amount of DNA synthesis. We can add this data redundantly for the experiment shown in Figure 3C to satisfy the point raised by the referee. Point 7 : We do not agree that the observation that Pol κ is required for the synthesis/stabilization of short replication intermediates is in discrepancy with the findings of Van et al., (JCB. 2010) since depletion of neither Pol δ nor Pol κ results in a complete disappearance of the short fragments (see Fig 5C-lane ΔPol δ/90 min in Van et al. and Fig 3C from our manuscript, lane 90 min), nor in a total decrease of P-Chk1 (see Fig 6B from Van et al and Fig 1A-B from our manuscript). Our observations show that, besides Pol δ, another polymerase is implicated at replication forks stalled after aphidicolin treatment, and underline the complexity of the system. Moreover, in Supplementary Figure 2A and Figure 3E of our manuscript we have shown that Pol k binds to chromatin in S-phase only in the presence of aphidicolin, which demonstrates that loading of Pol k occurs after that of Pol δ since Pol δ is already bound to chromatin in S-phase. We would like to stress that the aim of our

Background The molecular mechanisms involved in genetic instability, which is a driving force of cancer cells from earlier stages of pathologenesis, are not fully understood. Current evidence shows that overexpression of Pol θ, a “DNA repair” polymerase specialized in the replication of damaged DNA, which is altered in breast tumors, is not a passive agent in tumor development and is able to predict patient outcome. Aberrant POLQ expression may be related to genetic instability, and also resistance to “replicative stress”, leading to changes in replicating parameters and consequent tumor development. The objective of this project is to analyze genetic variants related to POLQ as new population biomarkers of risk, progression and prognosis in hereditary (HBC) and sporadic (SBC) breast cancer in Brazil.

Human DNA polymerase η (Pol η) is best known for its role in responding to UV irradiation-induced genome damage. We have recently observed that Pol η is also required for the stability of common fragile sites (CFSs), whose rearrangements are considered a driving force of oncogenesis. Here, we explored the molecular mechanisms underlying this newly identified role. We demonstrated that Pol η accumulated at CFSs upon partial replication stress and could efficiently replicate non-B DNA sequences within CFSs. Pol η deficiency led to persistence of checkpoint-blind under-replicated CFS regions in mitosis, detectable as FANCD2-associated chromosomal sites that were transmitted to daughter cells in 53BP1-shielded nuclear bodies. Expression of a catalytically inactive mutant of Pol η increased replication fork stalling and activated the replication checkpoint. These data are consistent with the requirement of Pol η-dependent DNA synthesis during S phase at replication forks stalled in CFS regions to suppress CFS instability by preventing checkpoint-blind under-replicated DNA in mitosis.

Specific DNA repair pathways from Trypanosoma cruzi are believed to protect genomic DNA and kinetoplast DNA (kDNA) from mutations. Particular pathways are supposed to operate in order to repair nucleotides oxidized by reactive oxygen species (ROS) during parasite infection, being 7,8-dihydro-8-oxoguanine (8oxoG) a frequent and highly mutagenic base alteration. If unrepaired, 8oxoG can lead to cytotoxic base transversions during DNA replication. In mammals, DNA polymerase beta (Polβ) is mainly involved in base excision repair (BER) of oxidative damage. However its biological role in T. cruzi is still unknown. We show, by immunofluorescence localization, that T. cruzi DNA polymerase beta (Tcpolβ) is restricted to the antipodal sites of kDNA in replicative epimastigote and amastigote developmental stages, being strictly localized to kDNA antipodal sites between G1/S and early G2 phase in replicative epimastigotes. Nevertheless, this polymerase was detected inside the mitochondrial matrix of trypomastigote forms, which are not able to replicate in culture. Parasites over expressing Tcpolβ showed reduced levels of 8oxoG in kDNA and an increased survival after treatment with hydrogen peroxide when compared to control cells. However, this resistance was lost after treating Tcpolβ overexpressors with methoxiamine, a potent BER inhibitor. Curiously, a presumed DNA repair focus containing Tcpolβ was identified in the vicinity of kDNA of cultured wild type epimastigotes after treatment with hydrogen peroxide. Taken together our data suggest participation of Tcpolβ during kDNA replication and repair of oxidative DNA damage induced by genotoxic stress in this organelle.

Lung cancer is the leading cause of cancer deaths worldwide. Clinical staging classification is generally insufficient to provide a reliable prognosis, particularly for early stages. In addition, prognostic factors are therefore needed to better forecast life expectancy and optimize adjuvant therapeutic strategy. Recent evidence indicates that alterations of the DNA replication program contribute to neoplasia from its early stages and that cancer cells are frequently exposed to endogenous replication stress. We therefore hypothesized that genes involved in the replication stress response may represent an under-explored source of biomarkers. Expressions of 77 DNA replication-associated genes implicated in different aspects of chromosomal DNA replication, including licensing, firing of origins, elongation, replication fork maintenance and recovery, lesion bypass and post-replicative repair were determined in primary tumors and adjacent normal tissues from 93 patients suffering from early- or mid-stage non-small cell lung cancer (NSCLC). We then investigated a statistically significant interaction between gene expressions and survival of early-stage NSCLC patients.The expression of five genes, that is, POLQ, PLK1, RAD51, CLASPIN and CDC6 was associated with overall, disease-free and relapse-free survival. The expression levels are independent of treatment and stage classification. Except RAD51, their prognostic role on survival persists after adjustment on age, sex, treatment, stage classification and conventional proliferation markers, with a hazard ratio of 36.3 for POLQ (95%CI 2.6–517.4, P=0.008), 23.5 for PLK1 (95%CI 1.9–288.4, P=0.01), 20.7 for CLASPIN (95%CI 1.5–275.9, P=0.02) and 18.5 for CDC6 (95%CI 1.3–267.4, P=0.03). We also show that a five-gene signature including POLQ, PLK1, RAD51, CLASPIN and CDC6 separates patients into low- and high-risk groups, with a hazard ratio of 14.3 (95% CI 5.1–40.3, P<0.001). This ‘replication stress’ metamarker may be a reliable predictor of survival for NSCLC, and may also help understand the molecular mechanisms underlying tumor progression.