RESUMEN
DNA mismatch repair (MMR) maintains genome stability through repair of DNA replication errors. In Escherichia coli, initiation of MMR involves recognition of the mismatch by MutS, recruitment of MutL, activation of endonuclease MutH and DNA strand incision at a hemimethylated GATC site. Here, we studied the mechanism of communication that couples mismatch recognition to daughter strand incision. We investigated the effect of catalytically-deficient Cas9 as well as stalled RNA polymerase as roadblocks placed on DNA in between the mismatch and GATC site in ensemble and single molecule nanomanipulation incision assays. The MMR proteins were observed to incise GATC sites beyond a roadblock, albeit with reduced efficiency. This residual incision is completely abolished upon shortening the disordered linker regions of MutL. These results indicate that roadblock bypass can be fully attributed to the long, disordered linker regions in MutL and establish that communication during MMR initiation occurs along the DNA backbone.
Asunto(s)
Reparación de la Incompatibilidad de ADN/genética , ADN Bacteriano/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas MutL/metabolismo , Disparidad de Par Base/genética , Proteína 9 Asociada a CRISPR/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Endodesoxirribonucleasas/metabolismo , Inestabilidad Genómica/genética , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismoRESUMEN
DNA mismatch repair (MMR) is an evolutionarily-conserved process responsible for the repair of replication errors. In Escherichia coli, MMR is initiated by MutS and MutL, which activate MutH to incise transiently-hemimethylated GATC sites. MMR efficiency depends on the distribution of these GATC sites. To understand which molecular events determine repair efficiency, we quantitatively studied the effect of strand incision on unwinding and excision activity. The distance between mismatch and GATC site did not influence the strand incision rate, and an increase in the number of sites enhanced incision only to a minor extent. Two GATC sites were incised by the same activated MMR complex in a processive manner, with MutS, the closed form of MutL and MutH displaying different roles. Unwinding and strand excision were more efficient on a substrate with two nicks flanking the mismatch, as compared to substrates containing a single nick or two nicks on the same side of the mismatch. Introduction of multiple nicks by the human MutLα endonuclease also contributed to increased repair efficiency. Our data support a general model of prokaryotic and eukaryotic MMR in which, despite mechanistic differences, mismatch-activated complexes facilitate efficient repair by creating multiple daughter strand nicks.
Asunto(s)
Reparación de la Incompatibilidad de ADN , Replicación del ADN , Disparidad de Par Base/genética , Secuencia de Bases , Metilación de ADN/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Conformación ProteicaRESUMEN
The endothelial to haematopoietic transition (EHT) is a key developmental process where a drastic change of endothelial cell morphology leads to the formation of blood stem and progenitor cells during embryogenesis. As TGFß signalling triggers a similar event during embryonic development called epithelial to mesenchymal transition (EMT), we hypothesised that TGFß activity could play a similar role in EHT as well. We used the mouse embryonic stem cell differentiation system for in vitro recapitulation of EHT and performed gain and loss of function analyses of the TGFß pathway. Quantitative proteomics analysis showed that TGFß treatment during EHT increased the secretion of several proteins linked to the vascular lineage. Live cell imaging showed that TGFß blocked the formation of round blood cells. Using gene expression profiling we demonstrated that the TGFß signalling activation decreased haematopoietic genes expression and increased the transcription of endothelial and extracellular matrix genes as well as EMT markers. Finally we found that the expression of the transcription factor Sox17 was up-regulated upon TGFß signalling activation and showed that its overexpression was enough to block blood cell formation. In conclusion we showed that triggering the TGFß pathway does not enhance EHT as we hypothesised but instead impairs it.
