Effect of monovalent cations and G-quadruplex structures on the outcome of intramolecular homologous recombination.
Homologous recombination is a very important cellular process, as it provides a major pathway for the repair of DNA double-strand breaks. This complex process is affected by many factors within cells. Here, we have studied the effect of monovalent cations (K+, Na+, and NH4+) on the outcome of recombination events, as their presence affects the biochemical activities of the proteins involved in recombination as well as the structure of DNA. For this purpose, we used an in vitro recombination system that includes a protein nuclear extract, as a source of recombination machinery, and two plasmids as substrates for intramolecular homologous recombination, each with two copies of different alleles of the human minisatellite MsH43. We found that the presence of monovalent cations induced a decrease in the recombination frequency, accompanied by an increase in the fidelity of the recombination. Moreover, there is an emerging consensus that secondary structures of DNA have the potential to induce genomic instability. Therefore, we analyzed the effect of the sequences capable of forming G-quadruplex on the production of recombinant molecules, taking advantage of the capacity of some MsH43 alleles to generate these kinds of structure in the presence of K+. We observed that the MsH43 recombinants containing duplications, generated in the presence of K+, did not include the repeats located towards the 5'-side of the G-quadruplex motif, suggesting that this structure may be involved in the recombination events leading to duplications. Our results provide new insights into the molecular mechanisms underlying the recombination of repetitive sequences.
Barros
Paula
P
Departamento de Bioquímica e Bioloxía Molecular, Facultade de Bioloxía-CIBUS, Universidade de Santiago de Compostela, Spain.
Boán
Francisco
F
Blanco
Miguel G
MG
Gómez-Márquez
Jaime
J
D000643:Ammonium Chloride / Q000494:pharmacology*; D002414:Cations, Monovalent / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel; D054856:G-Quadruplexes / Q000187:drug effects*; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics; D008957:Models, Genetic; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011189:Potassium Chloride / Q000494:pharmacology*; D011995:Recombination, Genetic / Q000187:drug effects*; D012965:Sodium Chloride / Q000494:pharmacology*
D002414:Cations, Monovalent; D000643:Ammonium Chloride; D012965:Sodium Chloride; D011189:Potassium Chloride