The LMO2 oncogene regulates DNA replication in hematopoietic cells.

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.

DNA polymerase ν gene expression influences fludarabine resistance in chronic lymphocytic leukemia independently of p53 status.

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).

DNA polymerase κ-dependent DNA synthesis at stalled replication forks is important for CHK1 activation.

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 DNA repair variant in POLQ (c.-1060A > G) is associated to hereditary breast cancer patients: a case-control study.

BACKGROUND: 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. METHODS: We analyzed through case-control study nine SNPs of POLQ in hereditary (HBC) and sporadic (SBC) breast cancer patients using Taqman Real Time PCR assays. Polymorphisms were systematically identified through the NCBI database and are located within exons or promoter regions. We recruited 204 breast cancer patients (101 SBC and 103 HBC) and 212 unaffected controls residing in Southern Brazil. RESULTS: The rs581553 SNP located in the promoter region was strongly associated with HBC (c.-1060A > G; HBC GG = 15, Control TT = 8; OR = 5.67, CI95% = 2.26-14.20; p < 0.0001). Interestingly, 11 of 15 homozygotes for this polymorphism fulfilled criteria for Hereditary Breast and Ovarian Cancer (HBOC) syndrome. Furthermore, 12 of them developed bilateral breast cancer and one had a familial history of bilateral breast cancer. This polymorphism was also associated with bilateral breast cancer in 67 patients (OR = 9.86, CI95% = 3.81-25.54). There was no statistically significant difference of age at breast cancer diagnosis between SNP carriers and non-carriers. CONCLUSIONS: Considering that Pol θ is involved in DBS repair, our results suggest that this polymorphism may contribute to the etiology of HBC, particularly in patients with bilateral breast cancer.

Impact of chromosomal instability on colorectal cancer progression and outcome.

BACKGROUND: 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). METHODS: In this work we set to determine the existence of such links between CRC progression and genetic instability and searched for associations with patient outcome. To this aim we analyzed a set of 162 Chromosomal Instable (CIN) CRCs comprising 131 primary carcinomas evenly distributed through stage 1 to 4, 31 metastases and 14 adenomas by array-CGH. CNA profiles were established according to disease stage and compared. We, also, asked whether the level of genomic instability was correlated to disease outcome in stage 2 and 3 CRCs. Two metrics of chromosomal instability were used; (i) Global Genomic Index (GGI), corresponding to the fraction of the genome involved in CNA, (ii) number of breakpoints (nbBP). RESULTS: Stage 1, 2, 3 and 4 tumors did not differ significantly at the level of their CNA profiles precluding the conventional definition of a progression scheme based on increasing levels of genetic instability. Combining GGI and nbBP,we classified genomic profiles into 5 groups presenting distinct patterns of chromosomal instability and defined two risk classes of tumors, showing strong differences in outcome and hazard risk (RFS: p = 0.012, HR = 3; OS: p < 0.001, HR = 9.7). While tumors of the high risk group were characterized by frequent fractional CNAs, low risk tumors presented predominantly whole chromosomal arm CNAs. Searching for CNAs correlating with negative outcome we found that losses at 16p13.3 and 19q13.3 observed in 10% (7/72) of stage 2-3 tumors showed strong association with early relapse (p < 0.001) and death (p < 0.007, p < 0.016). Both events showed frequent co-occurrence (p < 1x10-8) and could, therefore, mark for stage 2-3 CRC susceptible to negative outcome. CONCLUSIONS: Our data show that CRC disease progression from stage 1 to stage 4 is not paralleled by increased levels of genetic instability. However, they suggest that stage 2-3 CRC with elevated genetic instability and particularly profiles with fractional CNA represent a subset of aggressive tumors.

DNA polymerase theta up-regulation is associated with poor survival in breast cancer, perturbs DNA replication, and promotes genetic instability.

"Replicative stress" is one of the main factors underlying neoplasia from its early stages. Genes involved in DNA synthesis may therefore represent an underexplored source of potential prognostic markers for cancer. To this aim, we generated gene expression profiles from two independent cohorts (France, n=206; United Kingdom, n=117) of patients with previously untreated primary breast cancers. We report here that among the 13 human nuclear DNA polymerase genes, DNA Polymerase (POLQ) is the only one significantly up-regulated in breast cancer compared with normal breast tissues. Importantly, POLQ up-regulation significantly correlates with poor clinical outcome (4.3-fold increased risk of death in patients with high POLQ expression), and this correlation is independent of Cyclin E expression or the number of positive nodes, which are currently considered as markers for poor outcome. POLQ expression provides thus an additional indicator for the survival outcome of patients with high Cyclin E tumor expression or high number of positive lymph nodes. Furthermore, to decipher the molecular consequences of POLQ up-regulation in breast cancer, we generated human MRC5-SV cell lines that stably overexpress POLQ. Strong POLQ expression was directly associated with defective DNA replication fork progression and chromosomal damage. Therefore, POLQ overexpression may be a promising genetic instability and prognostic marker for breast cancer.

Aberrant expression of alternative DNA polymerases: a source of mutator phenotype as well as replicative stress in cancer.

The cell life span depends on a subtle equilibrium between the accurate duplication of the genomic DNA and less stringent DNA transactions which allow cells to tolerate mutations associated with DNA damage. The physiological role of the alternative, specialized or TLS (translesion synthesis) DNA polymerases could be to favor the necessary "flexibility" of the replication machinery, by allowing DNA replication to occur even in the presence of blocking DNA damage. As these alternative DNA polymerases are inaccurate when replicating undamaged DNA, the regulation of their expression needs to be carefully controlled. Evidence in the literature supports that dysregulation of these error-prone enzymes contributes to the acquisition of a mutator phenotype that, along with defective cell cycle control or other genome stability pathways, could be a motor for accelerated tumor progression.

The R438W polymorphism of human DNA polymerase lambda triggers cellular sensitivity to camptothecin by compromising the homologous recombination repair pathway.

The human DNA polymerase lambda (Polλ) is a DNA repair polymerase, which is believed not only to play a role in base excision repair but also to contribute to DNA double-strand break repair by non-homologous end joining. We described here that cellular expression of the recently described natural polymorphic variant of Polλ, Polλ(R438W), affects the homologous recombination (HR) pathway and sister chromatid exchange (SCE) events. We show that the HR defect provoked by this polymorphism enhances cellular sensitivity to the anticancer agent camptothecin (CPT), most of whose DNA damage is repaired by HR. All these effects were dependent on the DNA polymerase activity of Polλ(R438W) as the expression of a catalytically inactive Polλ(R438W) did not affect either the HR and SCE frequencies or the cellular sensitivity to CPT. These results suggest that sensitivity to CPT could result from cancer-related mutation in specialized DNA repair polymerases.

Biochemical studies with DNA polymerase beta and DNA polymerase beta-PAK of Trypanosoma cruzi suggest the involvement of these proteins in mitochondrial DNA maintenance.

Mammalian DNA polymerase beta is a nuclear enzyme involved in the base excision and single-stranded DNA break repair pathways. In trypanosomatids, this protein does not have a defined cellular localization, and its function is poorly understood. We characterized two Trypanosoma cruzi proteins homologous to mammalian DNA polymerasebeta, TcPolbeta and TcPolbetaPAK, and showed that both enzymes localize to the parasite kinetoplast. In vitro assays with purified proteins showed that they have DNA polymerization and deoxyribose phosphate lyase activities. Optimal conditions for polymerization were different for each protein with respect to dNTP concentration and temperature, and TcPolbetaPAK, in comparison to TcPolbeta, conducted DNA synthesis over a much broader pH range. TcPolbeta was unable to carry out mismatch extension or DNA synthesis across 8-oxodG lesions, and was able to discriminate between dNTP and ddNTP. These specific abilities of TcPolbeta were not observed for TcPolbetaPAK or other X family members, and are not due to a phenylalanine residue at position 395 in the C-terminal region of TcPolbeta, as assessed by a site-directed mutagenesis experiment reversing this residue to a well conserved tyrosine. Our data suggest that both polymerases from T. cruzi could cooperate to maintain mitochondrial DNA integrity through their multiple roles in base excision repair, gap filling and translesion synthesis.

Role of TLS DNA polymerases eta and kappa in processing naturally occurring structured DNA in human cells.

Accurate DNA replication during S-phase is fundamental to maintain genome integrity. During this critical process, replication forks frequently encounter obstacles that impede their progression. While the regulatory pathways which act in response to exogenous replication stress are beginning to emerge, the mechanisms by which fork integrity is maintained at naturally occurring endogenous replication-impeding sequences remains obscure. Notably, little is known about how cells replicate through special chromosomal regions containing structured non-B DNA, for example, G4 quartets, known to hamper fork progression or trigger chromosomal rearrangements. Here, we have investigated the role in this process of the human translesion synthesis (TLS) DNA polymerases of the Y-family (pol eta, pol iota, and pol kappa), specialized enzymes known to synthesize DNA through DNA damage. We show that depletion by RNA interference of expression of the genes for Pol eta or Pol kappa, but not Pol iota, sensitizes U2OS cells treated with the G4-tetraplex interactive compound telomestatin and triggers double-strand breaks in HeLa cells harboring multiple copies of a G-rich sequence from the promoter region of the human c-MYC gene, chromosomally integrated as a transgene. Moreover, we found that downregulation of Pol kappa only raises the level of DSB in HeLa cells containing either one of two breakage hotspot structured DNA sequences in the chromosome, the major break region (Mbr) of BCL-2 gene and the GA rich region from the far right-hand end of the genome of the Kaposi Sarcoma associated Herpesvirus. These data suggest that naturally occurring DNA structures are physiological substrates of both pol eta and pol kappa. We discuss these data in the light of their downregulation in human cancers.

Involvement of DNA polymerase mu in the repair of a specific subset of DNA double-strand breaks in mammalian cells.

The repair of DNA double-strand breaks (DSB) requires processing of the broken ends to complete the ligation process. Recently, it has been shown that DNA polymerase mu (polmu) and DNA polymerase lambda (pollambda) are both involved in such processing during non-homologous end joining in vitro. However, no phenotype was observed in animal models defective for either polmu and/or pollambda. Such observations could result from a functional redundancy shared by the X family of DNA polymerases. To avoid such redundancy and to clarify the role of polmu in the end joining process, we generated cells over-expressing the wild type as well as an inactive form of polmu (polmuD). We observed that cell sensitivity to ionizing radiation (IR) was increased when either polmu or polmuD was over-expressed. However, the genetic instability in response to IR increased only in cells expressing polmuD. Moreover, analysis of intrachromosomal repair of the I-SceI-induced DNA DSB, did not reveal any effect of either polmu or polmuD expression on the efficiency of ligation of both cohesive and partially complementary ends. Finally, the sequences of the repaired ends were specifically affected when polmu or polmuD was over-expressed, supporting the hypothesis that polmu could be involved in the repair of a DSB subset when resolution of junctions requires some gap filling.

Structural insights on the pamoic acid and the 8 kDa domain of DNA polymerase beta complex: towards the design of higher-affinity inhibitors.

BACKGROUND: DNA polymerase beta (pol beta), the error-prone DNA polymerase of single-stranded DNA break repair as well as base excision repair pathways, is overexpressed in several tumors and takes part in chemotherapeutic agent resistance, like that of cisplatin, through translesion synthesis. For this reason pol beta has become a therapeutic target. Several inhibitors have been identified, but none of them presents a sufficient affinity and specificity to become a drug. The fragment-based inhibitor design allows an important improvement in affinity of small molecules. The initial and critical step for setting up the fragment-based strategy consists in the identification and structural characterization of the first fragment bound to the target. RESULTS: We have performed docking studies of pamoic acid, a 9 micromolar pol beta inhibitor, and found that it binds in a single pocket at the surface of the 8 kDa domain of pol beta. However, docking studies provided five possible conformations for pamoic acid in this site. NMR experiments were performed on the complex to select a single conformation among the five retained. Chemical Shift Mapping data confirmed pamoic acid binding site found by docking while NOESY and saturation transfer experiments provided distances between pairs of protons from the pamoic acid and those of the 8 kDa domain that allowed the identification of the correct conformation. CONCLUSION: Combining NMR experiments on the complex with docking results allowed us to build a three-dimensional structural model. This model serves as the starting point for further structural studies aimed at improving the affinity of pamoic acid for binding to DNA polymerase beta.

The DNA polymerase lambda is required for the repair of non-compatible DNA double strand breaks by NHEJ in mammalian cells.

DNA polymerase lambda (pollambda) is a recently identified DNA polymerase whose cellular function remains elusive. Here we show, that pollambda participates at the molecular level in a chromosomal context, in the repair of DNA double strand breaks (DSB) via non-homologous end joining (NHEJ) in mammalian cells. The expression of a catalytically inactive form of pollambda (pollambdaDN) decreases the frequency of NHEJ events in response to I-Sce-I-induced DSB whereas inactivated forms of its homologues polbeta and polmu do not. Only events requiring DNA end processing before ligation are affected; this defect is associated with large deletions arising in the vicinity of the induced DSB. Furthermore, pollambdaDN-expressing cells exhibit increased sensitization and genomic instability in response to ionizing radiation similar to that of NHEJ-defective cells. Our data support a requirement for pollambda in repairing a subset of DSB in genomic DNA, thereby contributing to the maintenance of genetic stability mediated by the NHEJ pathway.

Up-regulation of the error-prone DNA polymerase {kappa} promotes pleiotropic genetic alterations and tumorigenesis.

It is currently widely accepted that genetic instability is key to cancer development. Many types of cancers arise as a consequence of a gradual accumulation of nucleotide aberrations, each mutation conferring growth and/or survival advantage. Genetic instability could also proceed in sudden bursts leading to a more drastic upheaval of structure and organization of the genome. Genetic instability, as an operative force, will produce genetic variants and the greater the instability, the larger the number of variants. We report here that the overexpression of human DNA polymerase kappa, an error-prone enzyme that is up-regulated in lung cancers, induces DNA breaks and stimulates DNA exchanges as well as aneuploidy. Probably as the result of so many perturbations, excess polymerase kappa favors the proliferation of competent tumor cells as observed in immunodeficient mice. These data suggest that altered regulation of DNA metabolism might be related to cancer-associated genetic changes and phenotype.

Human Adipose-Derived Stem Cells Expanded Under Ambient Oxygen Concentration Accumulate Oxidative DNA Lesions and Experience Procarcinogenic DNA Replication Stress.

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. Stem Cells Translational Medicine 2017;6:68-76.

Characterization of the Trypanosoma cruzi Rad51 gene and its role in recombination events associated with the parasite resistance to ionizing radiation.

The Rad51 gene encodes a highly conserved enzyme involved in DNA double-strand break (DSB) repair and recombination processes. We cloned and characterized the Rad51 gene from Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. This gene is expressed in all three forms of the parasite life cycle, with mRNA levels that are two-fold more abundant in the intracellular amastigote form. The recombinase activity of the TcRad51 gene product was verified by an increase in recombination events observed in transfected mammalian cells expressing TcRad51 and containing two inactive copies of the neomycin-resistant gene. As a component of the DSB repair machinery, we investigated the role of TcRad51 in the resistance to ionizing radiation and zeocin treatment presented by T. cruzi. When exposed to gamma irradiation, different strains of the parasite survive to dosages as high as 1 kGy. A role for TcRad51 in this process was evidenced by the increased expression of its mRNA after irradiation. Furthermore, transfected parasites over-expressing TcRad51 have a faster kinetics of recovery of the normal pattern of chromosomal bands after irradiation as well as a higher resistance to zeocin treatment than do wild-type cultures.

Enhanced expression and activity of DNA polymerase beta in chronic myelogenous leukemia.

BACKGROUND: Chronic myelogenous leukemia (CML) is characterized by an initial chronic phase that invariably evolves to the more aggressive phase of blast crisis. Although the determinants of this transition are still unknown, it has been shown that the blast crisis is accompanied by genetic instability. MATERIALS AND METHODS: The expression and activity of the error-prone DNA polymerase beta (pol beta) were investigated in blood samples from CML patients, by Western blotting and by an in vitro replication assay, respectively. RESULTS: Pol beta expression and activity were significantly higher in CML samples compared to those of healthy donors. CONCLUSION: Our results suggest that the excess of pol beta in CML could contribute to the genetic instability observed during the evolution of the disease from the chronic phase to blast crisis.

DNA polymerase beta overexpression stimulates the Rad51-dependent homologous recombination in mammalian cells.

Overexpression of DNA polymerase beta (polbeta), an error-prone DNA repair enzyme, has been shown to result in mutagenesis, aneuploidy and tumorigenesis. To further investigate the molecular basis leading to cancer-associated genetic changes, we examined whether the DNA polbeta could affect homologous recombination (HR). Using mammalian cells carrying an intrachromosomal recombination marker we showed that the DNA polbeta overexpression increased the HR mostly by enhancing gene conversion. Concomitantly, we observed the generation of DNA strand breaks as well as a DNA polbeta-dependent formation of Rad51 foci. The stimulation of HR was abolished by the coexpression of a dominant negative form of Rad51, suggesting that the Rad51 was involved in the increased HR events. The expression of different DNA polbeta mutants lacking polymerase activity did not result in HR stimulation, indicating that the DNA synthesis activity of DNA polbeta was related to this phenotype. These results provide new insights into the molecular mechanisms of the genetic instability observed in DNA polbeta overexpressing tumour cells.

Deregulated DNA polymerase beta induces chromosome instability and tumorigenesis.

To reach the biological alterations that characterize cancer, the genome of tumor cells must acquire increased mutability resulting from a malfunction of a network of genome stability systems, e.g., cell cycle arrest, DNA repair, and high accuracy of DNA synthesis during DNA replication. Numeric chromosomal imbalance, referred to as aneuploidy, is the most prevalent genetic changes recorded among many types of solid tumors. We report here that ectopic expression in cells of DNA polymerase beta, an error-prone enzyme frequently over-regulated in human tumors, induces aneuploidy, an abnormal localization of the centrosome-associated gamma-tubulin protein during mitosis, a deficient mitotic checkpoint, and promotes tumorigenesis in nude immunodeficient mice. Thus, we find that alteration of polymerase beta expression appears to induce major genetic changes associated with a malignant phenotype.

Cyclin Kinase-independent role of p21CDKN1A in the promotion of nascent DNA elongation in unstressed cells.

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.