RESUMO
The Mre11-Rad50-Nbs1 (MRN) complex is a central factor in the repair of DNA double-strand breaks (DSBs). The ATP-dependent mechanisms of how MRN detects and endonucleolytically processes DNA ends for the repair by microhomology-mediated end-joining or further resection in homologous recombination are still unclear. Here, we report the crystal structures of the ATPγS-bound dimer of the Rad50(NBD)(nucleotide-binding domain) from the thermophilic eukaryote Chaetomium thermophilum(Ct) in complex with either DNA or CtMre11(RBD)(Rad50-binding domain) along with small-angle X-ray scattering and cross-linking studies. The structure and DNA binding motifs were validated by DNA binding experiments in vitro and mutational analyses in Saccharomyces cerevisiae in vivo Our analyses provide a structural framework for the architecture of the eukaryotic Mre11-Rad50 complex. They show that a Rad50 dimer binds approximately 18 base pairs of DNA along the dimer interface in anATP-dependent fashion or bridges two DNA ends with a preference for 3' overhangs. Finally, our results may provide a general framework for the interaction of ABC ATPase domains of the Rad50/SMC/RecN protein family with DNA.
Assuntos
Trifosfato de Adenosina/metabolismo , Chaetomium/metabolismo , DNA Fúngico/metabolismo , Proteínas Fúngicas/metabolismo , Chaetomium/genética , Reparo do DNA , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Conformação ProteicaRESUMO
Together with the Rad50 ATPase, the Mre11 nuclease forms an evolutionarily conserved protein complex that plays a central role in the repair of DNA double-strand breaks (DSBs). Mre11-Rad50 detects and processes DNA ends, and has functions in the tethering as well as the signalling of DSBs. The Mre11 dimer can bind one or two DNA ends or hairpins, and processes DNA endonucleolytically as well as exonucleolytically in the 3'-to-5' direction. Here, the crystal structure of the Mre11 catalytic domain dimer from Chaetomium thermophilum (CtMre11(CD)) is reported. CtMre11(CD) crystals diffracted to 2.8 Å resolution and revealed previously undefined features within the dimer interface, in particular fully ordered eukaryote-specific insertion loops that considerably expand the dimer interface. Furthermore, comparison with other eukaryotic Mre11 structures reveals differences in the conformations of the dimer and the capping domain. In summary, the results reported here provide new insights into the architecture of the eukaryotic Mre11 dimer.
Assuntos
Chaetomium/química , Enzimas Reparadoras do DNA/química , Reparo do DNA , DNA/química , Proteínas Fúngicas/química , Motivos de Aminoácidos , Chaetomium/enzimologia , Chaetomium/genética , Cristalização , Cristalografia por Raios X , DNA/genética , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Expressão Gênica , Modelos Moleculares , Dados de Sequência Molecular , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homologia Estrutural de ProteínaRESUMO
DNA double-strand breaks are repaired by two major pathways, homologous recombination or nonhomologous end joining. The commitment to one or the other pathway proceeds via different steps of resection of the DNA ends, which is controlled and executed by a set of DNA double-strand break sensors, endo- and exonucleases, helicases, and DNA damage response factors. The molecular choreography of the underlying protein machinery is beginning to emerge. In this review, we discuss the early steps of genetic recombination and double-strand break sensing with an emphasis on structural and molecular studies.