RESUMO
Compared to humans, chimpanzees appear to be less susceptible to many types of cancer. Because DNA repair defects lead to accumulation of gene and chromosomal mutations, species differences in DNA repair are one plausible explanation. Here we analyzed the repair kinetics of human and chimpanzee cells after cisplatin treatment and irradiation. Dot blots for the quantification of single-stranded (ss) DNA repair intermediates revealed a biphasic response of human and chimpanzee lymphoblasts to cisplatin-induced damage. The early phase of DNA repair was identical in both species with a peak of ssDNA intermediates at 1 h after DNA damage induction. However, the late phase differed between species. Human cells showed a second peak of ssDNA intermediates at 6 h, chimpanzee cells at 5 h. One of four analyzed DNA repair-associated genes, UBE2A, was differentially expressed in human and chimpanzee cells at 5 h after cisplatin treatment. Immunofluorescent staining of gammaH2AX foci demonstrated equally high numbers of DNA strand breaks in human and chimpanzee cells at 30 min after irradiation and equally low numbers at 2 h. However, at 1 h chimpanzee cells had significantly less DNA breaks than human cells. Comparative sequence analyses of approximately 100 DNA repair-associated genes in human and chimpanzee revealed 13% and 32% genes, respectively, with evidence for an accelerated evolution in promoter regions and introns. This is strikingly contrasting to the 3% of DNA repair-associated genes with positive selection in the coding sequence. Compared to the rhesus macaque as an outgroup, chimpanzees have a higher accelerated evolution in non-coding sequences than humans. The TRF1-interacting, ankyrin-related ADP-ribose polymerase (TNKS) gene showed an accelerated intraspecific evolution among humans. Our results are consistent with the view that chimpanzee cells repair different types of DNA damage faster than human cells, whereas the overall repair capacity is similar in both species. Genetic differences in non-coding sequence elements may affect gene regulation in the DNA repair network and thus contribute to species differences in DNA repair and cancer susceptibility.
Assuntos
Dano ao DNA/genética , Reparo do DNA/genética , DNA/genética , Pan troglodytes/genética , Animais , Sequência de Bases , Células Cultivadas , Cisplatino/farmacologia , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/efeitos da radiação , RNA Mensageiro/genéticaRESUMO
Fanconi anemia (FA) cells are generally hypersensitive to DNA cross-linking agents, implying that mutations in the different FANC genes cause a similar DNA repair defect(s). By using a customized cDNA microarray chip for DNA repair- and cell cycle-associated genes, we identified three genes, cathepsin B (CTSB), glutaredoxin (GLRX), and polo-like kinase 2 (PLK2), that were misregulated in untreated primary fibroblasts from three unrelated FA-D2 patients, compared to six controls. Quantitative real-time RT PCR was used to validate these results and to study possible molecular links between FA-D2 and other FA subtypes. GLRX was misregulated to opposite directions in a variety of different FA subtypes. Increased CTSB and decreased PLK2 expression was found in all or almost all of the analyzed complementation groups and, therefore, may be related to the defective FA pathway. Transcriptional upregulation of the CTSB proteinase appears to be a secondary phenomenon due to proliferation differences between FA and normal fibroblast cultures. In contrast, PLK2 is known to play a pivotal role in processes that are linked to FA defects and may contribute in multiple ways to the FA phenotype: PLK2 is a target gene for TP53, is likely to function as a tumor suppressor gene in hematologic neoplasia, and Plk2(-/-) mice are small because of defective embryonal development.
Assuntos
Anemia de Fanconi/genética , RNA Mensageiro/genética , Estudos de Casos e Controles , Catepsina B/genética , Ciclo Celular/genética , Citogenética , Reparo do DNA/genética , Anemia de Fanconi/classificação , Anemia de Fanconi/metabolismo , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Glutarredoxinas/genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The RNA-dependent RNA polymerase (RdRp) of Tomato bushy stunt virus (TBSV) contains an arginine- and proline-rich (RPR) motif. This motif functions as an RNA-binding domain and is essential for tombusvirus replication. A mutant carrying three arginine substitutions in this motif rendered the virus unable to replicate in Nicotiana benthamiana plants and protoplasts. When the replicase function was provided in trans, by expressing the TBSV RdRp in N. benthamiana plants, an infectious variant could be isolated. Sequence analysis showed that only the substituted glycine residue (position 216) had reverted to arginine; all other substitutions remained unchanged. This finding suggested that strong selection pressure is active to maintain necessary sequences of the viral RdRp and that the analysis of revertants may help to identify essential viral functions.