RESUMO
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.
Assuntos
Reparo de Erro de Pareamento de DNA/genética , DNA Bacteriano/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas MutL/metabolismo , Pareamento Incorreto de Bases/genética , Proteína 9 Associada à CRISPR/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Endodesoxirribonucleases/metabolismo , Instabilidade Genômica/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismoRESUMO
In this issue of Structure, Cho and colleagues provide intriguing insight into the first steps of the DNA mismatch repair process. By using single-molecule techniques, they show that the protein MutS undergoes two different types of diffusion on error-containing DNA in an ATP-dependent way.
Assuntos
Trifosfato de Adenosina/química , Proteínas de Bactérias/química , DNA/química , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Thermus/químicaAssuntos
DNA Helicases/química , DNA Helicases/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Cromossomos Bacterianos , Escherichia coli/enzimologia , Escherichia coli/genética , Estrutura Terciária de ProteínaRESUMO
Single-molecule experiments show that the chromatin-remodeling complex RSC, a member of the SNF2 ATPase family, induces formation of a negatively supercoiled DNA loop by active translocation.