ABSTRACT
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.
Subject(s)
DNA/metabolism , Deoxyribonucleases/metabolism , Escherichia coli Proteins/metabolism , Exonucleases/metabolism , Adenosine Triphosphate/metabolism , DNA/chemistry , Deoxyribonucleases/chemistry , Deoxyribonucleases/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Exonucleases/chemistry , Exonucleases/genetics , Fluorescence Polarization , Hydrolysis , Phosphates/chemistry , Phosphates/metabolism , Protein MultimerizationABSTRACT
The HerA-NurA helicase-nuclease complex cooperates with Mre11 and Rad50 to coordinate the repair of double-stranded DNA breaks. Little is known, however, about the assembly mechanism and activation of the HerA-NurA. By combining hybrid mass spectrometry with cryo-EM, computational and biochemical data, we investigate the oligomeric formation of HerA and detail the mechanism of nucleotide binding to the HerA-NurA complex from thermophilic archaea. We reveal that ATP-free HerA and HerA-DNA complexes predominantly exist in solution as a heptamer and act as a DNA loading intermediate. The binding of either NurA or ATP stabilizes the hexameric HerA, indicating that HerA-NurA is activated by substrates and complex assembly. To examine the role of ATP in DNA translocation and processing, we investigated how nucleotides interact with the HerA-NurA. We show that while the hexameric HerA binds six nucleotides in an 'all-or-none' fashion, HerA-NurA harbors a highly coordinated pairwise binding mechanism and enables the translocation and processing of double-stranded DNA. Using molecular dynamics simulations, we reveal novel inter-residue interactions between the external ATP and the internal DNA binding sites. Overall, here we propose a stepwise assembly mechanism detailing the synergistic activation of HerA-NurA by ATP, which allows efficient processing of double-stranded DNA.
Subject(s)
Archaeal Proteins/metabolism , DNA Helicases/metabolism , DNA, Archaeal/metabolism , Deoxyribonucleases/metabolism , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Archaeal Proteins/chemistry , Archaeal Proteins/genetics , Binding Sites/genetics , DNA Breaks, Double-Stranded , DNA Helicases/chemistry , DNA Helicases/genetics , DNA Repair , DNA, Archaeal/chemistry , DNA, Archaeal/genetics , Deoxyribonucleases/chemistry , Deoxyribonucleases/genetics , Models, Molecular , Nucleic Acid Conformation , Protein Binding , Protein Domains , Protein Multimerization , Sulfolobus solfataricus/enzymology , Sulfolobus solfataricus/genetics , Sulfolobus solfataricus/metabolismABSTRACT
DNA double-strand breaks can be repaired by homologous recombination, during which the DNA ends are long-range resected by helicase-nuclease systems to generate 3' single strand tails. In archaea, this requires the Mre11-Rad50 complex and the ATP-dependent helicase-nuclease complex HerA-NurA. We report the cryo-EM structure of Sulfolobus solfataricus HerA-NurA at 7.4Å resolution and present the pseudo-atomic model of the complex. HerA forms an ASCE hexamer that tightly interacts with a NurA dimer, with each NurA protomer binding three adjacent HerA HAS domains. Entry to NurA's nuclease active sites requires dsDNA to pass through a 23Å wide channel in the HerA hexamer. The structure suggests that HerA is a dsDNA translocase that feeds DNA into the NurA nuclease sites.
