Structural basis for translocation by AddAB helicase–nuclease and its arrest at χ sites

Abstract

In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is dependent upon the recombination hotspot sequence χ (Chi)1,2 and is catalysed by either an AddAB- or RecBCD-type helicase–nuclease (reviewed in refs 3, 4). These enzyme complexes unwind and digest the DNA duplex from the broken end until they encounter a χ sequence5, whereupon they produce a 3′ single-stranded DNA tail onto which they initiate loading of the RecA protein6. Consequently, regulation of the AddAB/RecBCD complex by χ is a key control point in DNA repair and other processes involving genetic recombination. Here we report crystal structures of Bacillus subtilis AddAB in complex with different χ-containing DNA substrates either with or without a non-hydrolysable ATP analogue. Comparison of these structures suggests a mechanism for DNA translocation and unwinding, suggests how the enzyme binds specifically to χ sequences, and explains how χ recognition leads to the arrest of AddAB (and RecBCD) translocation that is observed in single-molecule experiments7,8,9.

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Figure 1: Structure of the binary and ternary complexes.
Figure 2: Proposed mechanism of unwinding and translocation.
Figure 3: The complex of AddAB bound to χ.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates have been deposited at the PDB with accession codes 4CEH, 4CEI and 4CEJ.

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Acknowledgements

We thank the European Synchrotron Radiation Facility and Diamond synchrotrons for access to beamlines. The work was funded by the Royal Society, the Wellcome Trust and the European Research Council (M.S.D.), Cancer Research UK (D.B.W.) and EMBO (W.W.K.).

Author information

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Authors

Contributions

W.W.K. and D.B.W. designed the experiments. W.W.K., X.F., M.W. and N.B.C. performed the experiments. W.W.K., M.W., M.S.D. and D.B.W. analysed the data and prepared the manuscript.

Corresponding author

Correspondence to Dale B. Wigley.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 DNA substrates used in the structure determinations.

We have previously determined the crystal structure of an initiation complex of AddAB using the DNA substrate shown at the top10. The substrates used in the current study differ by the additions shown in blue below. Five T residues were added to make a 5′ tail for the fork. The 3′ tail was extended by the addition of an AddAB χ sequence (AGCGG), preceded by a ‘spacer’ sequence of between 1 and 7 T residues to extend the distance between the fork junction and the χ sequence along the 3′ tail to mimic the products of sequential unwinding of a DNA fork. Oligo 023 used in this study contained a six-base T spacer. Oligo 027 contained a seven-T spacer plus an additional four T residues beyond the χ sequence as shown above. AddAB failed to recognize χ in substrates with six or less residues in the spacer but was able to bind to χ with seven T residues in the spacer region.

Extended Data Figure 2 Cartoon representation of the DNA-binding site in the two complexes.

Residues from AddA in orange, AddB in cyan, hydrogen bonds and charged interactions as arrows, hydrophobic and stacked interactions as solid lines.

Extended Data Figure 3 Nucleotide-binding sites.

Difference electron density (Fo – Fc, contoured at 2.5σ) for bound nucleotide at the ATP-binding sites in the AddA and AddB subunits in the ADPNP and χ complexes.

Extended Data Figure 4 Conformational variability of domain 2A of AddA in different χ-bound structures.

The two extreme variants are shown for illustration. Although domain 1A (green and light green) of the AddA protein (orange) remains in an essentially constant position, the position of the 2A domain (red and light red) varies from one that is almost superimposable with the ‘apo’ complex that lacks ADPNP, to one that is midway between the apo and ADPNP-bound conformations plus a variety of conformations between these two extreme cases. The extreme cases differ by a rotation of 5° compared to 7° for the difference between the ADPNP and apo structures. By contrast, the conformation of the 2A domain of AddA adopts an almost invariable conformation in different structures of the AddAB–DNA complex grown with ADPNP with a variety of DNA substrates in which χ is not bound.

Extended Data Figure 5 Cartoon summarizing our current knowledge of the molecular structures of different functional states along the AddAB reaction pathway.

We now have crystal structures for the first three functional states as shown in the figure. These show how the protein complex interacts with DNA (initiation complex), what happens when ATP binds and how this leads to DNA unwinding (translocation complex) and what happens when this complex encounters a χ sequence (χ recognition complex). At least two further states have yet to be visualized: (1) what happens after the pause at χ (χ reactivation complex) and (2) how the protein interacts with and loads RecA (RecA-loading complex).

Extended Data Table 1 X-ray data and refinement statistics

Supplementary information

DNA translocation mechanism

This video depicts a morph between the binary and ternary complexes. The details are described in the main text. Note that this is likely a simplification for the mechanism which assumes just two states and additional states on the unwinding pathway may well exist. The video shows two cycles of ATP binding and hydrolysis. (MOV 3591 kb)

Translocation mechanism emphasising the role of the motor domains of AddA

Similar to Supplementary video 1, although this video emphasises the role of the AddA domains in the unwinding mechanism. The 1A domain is shown in green, 2A in red and 1B in blue. The remainder of the protein is shown translucent orange (AddA) or cyan (AddB) for context. (MOV 3554 kb)

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Krajewski, W., Fu, X., Wilkinson, M. et al. Structural basis for translocation by AddAB helicase–nuclease and its arrest at χ sites. Nature 508, 416–419 (2014). https://doi.org/10.1038/nature13037

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