ABSTRACT
In most bacteria, cell division relies on the synthesis of new cell wall material by the multiprotein divisome complex. Thus, at the core of the divisome are the transglycosylase FtsW, which synthesises peptidoglycan strands from its substrate Lipid II, and the transpeptidase FtsI that cross-links these strands to form a mesh, shaping and protecting the bacterial cell. The FtsQ-FtsB-FtsL trimeric complex interacts with the FtsWI complex and is involved in regulating its enzymatic activities; however, the structure of this pentameric complex is unknown. Here, we present the cryogenic electron microscopy structure of the FtsWIQBL complex from Pseudomonas aeruginosa at 3.7 Å resolution. Our work reveals intricate structural details, including an extended coiled coil formed by FtsL and FtsB and the periplasmic interaction site between FtsL and FtsI. Our structure explains the consequences of previously reported mutations and we postulate a possible activation mechanism involving a large conformational change in the periplasmic domain. As FtsWIQBL is central to the divisome, our structure is foundational for the design of future experiments elucidating the precise mechanism of bacterial cell division, an important antibiotic target.
Subject(s)
Escherichia coli Proteins , Escherichia coli Proteins/genetics , Escherichia coli/physiology , Cell Cycle Proteins/genetics , Cryoelectron Microscopy , Membrane Proteins/geneticsABSTRACT
DNA double-strand breaks (DSBs) threaten genome stability and are linked to tumorigenesis in humans. Repair of DSBs requires the removal of attached proteins and hairpins through a poorly understood but physiologically critical endonuclease activity by the Mre11-Rad50 complex. Here, we report cryoelectron microscopy (cryo-EM) structures of the bacterial Mre11-Rad50 homolog SbcCD bound to a protein-blocked DNA end and a DNA hairpin. The structures reveal that Mre11-Rad50 bends internal DNA for endonucleolytic cleavage and show how internal DNA, DNA ends, and hairpins are processed through a similar ATP-regulated conformational state. Furthermore, Mre11-Rad50 is loaded onto blocked DNA ends with Mre11 pointing away from the block, explaining the distinct biochemistries of 3' â 5' exonucleolytic and endonucleolytic incision through the way Mre11-Rad50 interacts with diverse DNA ends. In summary, our results unify Mre11-Rad50's enigmatic nuclease diversity within a single structural framework and reveal how blocked DNA ends and hairpins are processed.
Subject(s)
DNA-Binding Proteins , DNA , MRE11 Homologue Protein/chemistry , Acid Anhydride Hydrolases/genetics , Acid Anhydride Hydrolases/metabolism , Adenosine Triphosphate/metabolism , Cryoelectron Microscopy , DNA/metabolism , DNA Repair , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Endonucleases/genetics , Exodeoxyribonucleases/genetics , Exodeoxyribonucleases/metabolism , Humans , Nucleic Acid ConformationABSTRACT
DNA double-strand breaks (DSBs) threaten genome stability throughout life and are linked to tumorigenesis in humans. To initiate DSB repair by end joining or homologous recombination, the Mre11-nuclease Rad50-ATPase complex detects and processes diverse and obstructed DNA ends, but a structural mechanism is still lacking. Here we report cryo-EM structures of the E. coli Mre11-Rad50 homolog SbcCD in resting and DNA-bound cutting states. In the resting state, Mre11's nuclease is blocked by ATP-Rad50, and the Rad50 coiled coils appear flexible. Upon DNA binding, the two coiled coils zip up into a rod and, together with the Rad50 nucleotide-binding domains, form a clamp around dsDNA. Mre11 moves to the side of Rad50, binds the DNA end, and assembles a DNA cutting channel for the nuclease reactions. The structures reveal how Mre11-Rad50 can detect and process diverse DNA ends and uncover a clamping and gating function for the coiled coils.
Subject(s)
Acid Anhydride Hydrolases/metabolism , DNA Breaks, Double-Stranded , DNA Replication , DNA, Bacterial/metabolism , Deoxyribonucleases/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Exonucleases/metabolism , MRE11 Homologue Protein/metabolism , Acid Anhydride Hydrolases/genetics , Acid Anhydride Hydrolases/ultrastructure , Cryoelectron Microscopy , DNA, Bacterial/genetics , DNA, Bacterial/ultrastructure , Deoxyribonucleases/genetics , Deoxyribonucleases/ultrastructure , Escherichia coli/genetics , Escherichia coli/ultrastructure , Escherichia coli Proteins/genetics , Escherichia coli Proteins/ultrastructure , Exonucleases/genetics , Exonucleases/ultrastructure , MRE11 Homologue Protein/genetics , MRE11 Homologue Protein/ultrastructure , Nucleic Acid Conformation , Structure-Activity RelationshipABSTRACT
The Mre11-Rad50 complex is a DNA double-strand break sensor that cleaves blocked DNA ends and hairpins by an ATP-dependent endo/exonuclease activity for subsequent repair. For that, Mre11-Rad50 complexes, including the Escherichia coli homolog SbcCD, can endonucleolytically cleave one or both strands near a protein block and process free DNA ends via a 3'-5' exonuclease, but a unified basis for these distinct activities is lacking. Here we analyzed DNA binding, ATPase and nuclease reactions on different DNA substrates. SbcCD clips terminal bases of both strands of the DNA end in the presence of ATPγS. It introduces a DNA double-strand break around 20-25 bp from a blocked end after multiple rounds of ATP hydrolysis in a reaction that correlates with local DNA meltability. Interestingly, we find that nuclease reactions on opposing strands are chemically distinct, leaving a 5' phosphate on one strand, but a 3' phosphate on the other strand. Collectively, our results identify an unexpected chemical variability of the nuclease, indicating that the complex is oriented at a free DNA end and facing a block with opposite polarity. This suggests a unified model for ATP-dependent endo- and exonuclease reactions at internal DNA near a block and at free DNA ends.
