RESUMO
A newly identified process by which mistargeted V(D)J recombination could cause genome instability in childhood leukemia has been discovered. In this mechanism, called cut-and-run, the excised DNA by-products of V(D)J recombination are re-bound by the recombinase proteins and erroneously trigger double-strand breaks at multiple locations throughout the genome. Many of these breakpoints co-localize with known chromosome alterations in acute lymphoblastic leukemia (ALL).
RESUMO
V(D)J recombination is essential to generate antigen receptor diversity but is also a potent cause of genome instability. Many chromosome alterations that result from aberrant V(D)J recombination involve breaks at single recombination signal sequences (RSSs). A long-standing question, however, is how such breaks occur. Here, we show that the genomic DNA that is excised during recombination, the excised signal circle (ESC), forms a complex with the recombinase proteins to efficiently catalyze breaks at single RSSs both in vitro and in vivo. Following cutting, the RSS is released while the ESC-recombinase complex remains intact to potentially trigger breaks at further RSSs. Consistent with this, chromosome breaks at RSSs increase markedly in the presence of the ESC. Notably, these breaks co-localize with those found in acute lymphoblastic leukemia patients and occur at key cancer driver genes. We have named this reaction "cut-and-run" and suggest that it could be a significant cause of lymphocyte genome instability.
Assuntos
Instabilidade Genômica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocação Genética/genética , Recombinação V(D)J/genética , Animais , Sequência de Bases/genética , Células COS , Chlorocebus aethiops , Cromossomos/genética , DNA/genética , Quebras de DNA de Cadeia Dupla , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Células NIH 3T3 , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Recombinases/genéticaRESUMO
TALE (transcription activator-like effector) proteins can be tailored to bind to any DNA sequence of choice and thus are of immense utility for genome editing and the specific delivery of transcription activators. However, to perform these functions, they need to occupy their sites in chromatin. In the present study, we have systematically assessed TALE binding to chromatin substrates and find that in vitro TALEs bind to their target site on nucleosomes at the more accessible entry/exit sites, but not at the nucleosome dyad. We show further that in vivo TALEs bind to transcriptionally repressed chromatin and that transcription increases binding by only 2-fold. These data therefore imply that TALEs are likely to bind to their target in vivo even at inactive loci.
