Drosophila DNA polymerase theta utilizes both helicase-like and polymerase domains during microhomology-mediated end joining and interstrand crosslink repair

Double strand breaks (DSBs) and interstrand crosslinks (ICLs) are toxic DNA lesions that can be repaired through multiple pathways, some of which involve shared proteins. One of these proteins, DNA Polymerase theta (Pol theta), coordinates a mutagenic DSB repair pathway named microhomology-mediated end joining (MMEJ) and is also a critical component for bypass or repair of ICLs in several organisms. Pol theta contains both polymerase and helicase-like domains that are tethered by an unstructured central region. While the role of the polymerase domain in promoting MMEJ has been studied extensively both in vitro and in vivo, a function for the helicase-like domain, which possesses DNA-dependent ATPase activity, remains unclear. Here, we utilize genetic and biochemical analyses to examine the roles of the helicase-like and polymerase domains of Drosophila Pol theta. We demonstrate an absolute requirement for both polymerase and ATPase activities during ICL repair in vivo. However, similar to mammalian systems, polymerase activity, but not ATPase activity, is required for ionizing radiation-induced DSB repair. Using a site-specific break repair assay, we show that overall end-joining efficiency is not affected in ATPase-dead mutants, but there is a significant decrease in templated insertion events. In vitro, Pol theta can efficiently bypass a model unhooked nitrogen mustard crosslink and promote DNA synthesis following microhomology annealing, although ATPase activity is not required for these functions. Together, our data illustrate the functional importance of the helicase-like domain of Pol theta and suggest that its tethering to the polymerase domain is important for its multiple functions in DNA repair and damage tolerance

DNA replication stress response involving PLK1, CDC6, POLQ, RAD51 and CLASPIN upregulation prognoses the outcome of early/mid-stage non-small cell lung cancer patients

International audienceLung cancer is the leading cause of cancer deaths worldwide. Clinical staging classification is generally insufficient to providea reliable prognosis, particularly for early stages. In addition, prognostic factors are therefore needed to better forecast lifeexpectancy and optimize adjuvant therapeutic strategy. Recent evidence indicates that alterations of the DNA replication programcontribute 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 ofbiomarkers. 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 repairwere determined in primary tumors and adjacent normal tissues from 93 patients suffering from early- or mid-stage non-small celllung 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, CLASPINandCDC6was associated with overall,disease-free and relapse-free survival. The expression levels are independent of treatment and stage classification. ExceptRAD51,their prognostic role on survival persists after adjustment on age, sex, treatment, stage classification and conventional proliferationmarkers, with a hazard ratio of 36.3 forPOLQ(95%CI 2.6–517.4,P¼0.008), 23.5 forPLK1(95%CI 1.9–288.4,P¼0.01), 20.7 forCLASPIN(95%CI 1.5–275.9,P¼0.02) and 18.5 forCDC6(95%CI 1.3–267.4,P¼0.03). We also show that a five-gene signature includingPOLQ,PLK1, RAD51, CLASPINandCDC6separates patients into low- and high-risk groups, with a hazard ratio of 14.3 (95% CI 5.1–40.3,Po0.001). This ‘replication stress’ metamarker may be a reliable predictor of survival for NSCLC, and may also help understand themolecular mechanisms underlying tumor progression

Efficient gene transfer in skeletal muscle with AAV-derived bicistronic vector using the FGF-1 IRES.

International audienceIRESs (internal ribosome entry sites) are RNA elements behaving as translational enhancers in conditions of global translation blockade. IRESs are also useful in biotechnological applications as they allow expression of several genes from a single mRNA. Up to now, most IRES-containing vectors use the IRES from encephalomyocarditis virus (EMCV), highly active in transiently transfected cells but long and not flexible in its positioning relative to the gene of interest. In contrast, several IRESs identified in cellular mRNAs are short and flexible and may therefore be advantageous in gene transfer vectors such as those derived from the adeno-associated virus (AAV), where the size of the transgene expression cassette is limited. Here, we have tested bicistronic AAV-derived vectors expressing two luciferase genes separated by the EMCV- or fibroblast growth factor 1 (FGF-1) IRES. We demonstrate that the AAV vector with the FGF-1 IRES, when administrated into the mouse muscle, leads to efficient expression of both transgenes with a stable stoechiometry, for at least 120 days. Interestingly, the bicistronic mRNA containing the FGF-1 IRES leads to transgene expression 10 times superior to that observed with EMCV, in vivo. AAV vectors featuring the FGF-1 IRES may thus be advantageous for gene therapy approaches in skeletal muscle involving coexpression of genes of interest

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