RESUMO
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.
Assuntos
Divisão Celular , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Replicação do DNA , DNA/biossíntese , Instabilidade Genômica , Células Cultivadas , HumanosRESUMO
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.
Assuntos
Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , Predisposição Genética para Doença , Variação Genética , Alelos , Substituição de Aminoácidos , Neoplasias da Mama/genética , Estudos de Casos e Controles , Feminino , Frequência do Gene , Genótipo , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Humanos , Razão de Chances , Polimorfismo de Nucleotídeo Único , DNA Polimerase tetaRESUMO
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 (Polß) 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 (Tcpolß) 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 Tcpolß 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 Tcpolß overexpressors with methoxiamine, a potent BER inhibitor. Curiously, a presumed DNA repair focus containing Tcpolß was identified in the vicinity of kDNA of cultured wild type epimastigotes after treatment with hydrogen peroxide. Taken together our data suggest participation of Tcpolß during kDNA replication and repair of oxidative DNA damage induced by genotoxic stress in this organelle.
Assuntos
DNA Polimerase beta/metabolismo , Reparo do DNA , Replicação do DNA , DNA de Cinetoplasto/metabolismo , Trypanosoma cruzi/enzimologia , Microscopia de Fluorescência , Mitocôndrias/química , Mitocôndrias/enzimologia , Estresse Oxidativo , Trypanosoma cruzi/química , Trypanosoma cruzi/genéticaRESUMO
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.
Assuntos
DNA Polimerase beta/metabolismo , DNA Mitocondrial/metabolismo , Trypanosoma cruzi/enzimologia , Quinases Ativadas por p21/metabolismo , Sequência de Aminoácidos , Animais , Bioquímica/métodos , Clonagem Molecular , Primers do DNA/química , Microscopia Confocal , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Genes de Protozoários , Proteínas de Protozoários/genética , Rad51 Recombinase/genética , Trypanosoma cruzi/genética , Trypanosoma cruzi/efeitos da radiação , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Cricetinae , DNA de Protozoário/genética , Raios gama , Humanos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Tolerância a Radiação/genética , Recombinação Genética , Homologia de Sequência de Aminoácidos , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/patogenicidadeRESUMO
Five new acylphloroglucinol derivatives, mahureones A-E (1, 3-6), have been isolated from the leaves of Mahurea palustris, and their structures determined by spectroscopic means. During the isolation process, several byproducts (7-9) were formed by reaction of one of the isoprenyl side chains with TFA, water, and acetonitrile. All the compounds were assayed for their ability to inhibit human DNA polymerase beta. The most active compounds, mahureones A (1) and D (5), exhibited IC50 values in the 10 microM range.