Structural mechanism of ATP-dependent DNA binding and DNA end bridging by eukaryotic Rad50

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 ATPS-bound dimer of the Rad50NBD (nucleotide-binding domain) from the thermophilic eukaryote Chaetomium thermophilum (Ct) in complex with either DNA or CtMre11RBD (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 an ATP-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.
Source: EMBO Journal - Category: Molecular Biology Authors: Tags: DNA Replication, Repair & Recombination, Structural Biology Articles Source Type: research

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We present a reproducible knock-in strategy combined with a highly specific homologous directed repair (HDR) screening approach using digital droplet PCR (ddPCR). The knock-in cell lines that we created show a functioning fluorescent reporter system for DA neurons that are identifiable by flow cytometry.
Source: Stem Cell Research - Category: Stem Cells Source Type: research
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Source: Journal of Materials Research and Technology - Category: Materials Science Source Type: research
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Source: PediatricEducation.org - Category: Pediatrics Authors: Tags: Uncategorized Source Type: news
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Source: European Journal of Pediatrics - Category: Pediatrics Source Type: research
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Source: Molecular Medicine Reports - Category: Molecular Biology Tags: Mol Med Rep Source Type: research
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Source: European Journal of Obstetrics and Gynecology and Reproductive Biology - Category: OBGYN Source Type: research
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Source: Fight Aging! - Category: Research Authors: Tags: Newsletters Source Type: blogs
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Source: Mol Biol Cell - Category: Molecular Biology Authors: Tags: Methods Mol Biol Source Type: research
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Source: Photochemistry and Photobiology - Category: Science Authors: Tags: Research Article Source Type: research
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