Structures of RecBCD in complex with phage-encoded inhibitor proteins reveal distinctive strategies for evasion of a bacterial immunity hub
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eLife assessment
This important study addresses the ways in which bacteriophages antagonize or coopt the DNA restriction and/or recombination functions of the bacterial RecBCD helicase-nuclease. The evidence from both biochemistry and structural biology showing convergent evolution is convincing.
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Abstract
Following infection of bacterial cells, bacteriophage modulate double-stranded DNA break repair pathways to protect themselves from host immunity systems and prioritise their own recombinases. Here, we present biochemical and structural analysis of two phage proteins, gp5.9 and Abc2, which target the DNA break resection complex RecBCD. These exemplify two contrasting mechanisms for control of DNA break repair in which the RecBCD complex is either inhibited or co-opted for the benefit of the invading phage. Gp5.9 completely inhibits RecBCD by preventing it from binding to DNA. The RecBCD-gp5.9 structure shows that gp5.9 acts by substrate mimicry, binding predominantly to the RecB arm domain and competing sterically for the DNA binding site. Gp5.9 adopts a parallel coiled-coil architecture that is unprecedented for a natural DNA mimic protein. In contrast, binding of Abc2 does not substantially affect the biochemical activities of isolated RecBCD. The RecBCD-Abc2 structure shows that Abc2 binds to the Chi-recognition domains of the RecC subunit in a position that might enable it to mediate the loading of phage recombinases onto its single-stranded DNA products.
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eLife assessment
This important study addresses the ways in which bacteriophages antagonize or coopt the DNA restriction and/or recombination functions of the bacterial RecBCD helicase-nuclease. The evidence from both biochemistry and structural biology showing convergent evolution is convincing.
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Reviewer #1 (Public Review):
This paper provides biochemical and structural evidence for how two different phage proteins inhibit the RecBCD system. The paper provides interesting new insights into the battle that takes place between bacteria and phages and shows how convergent evolution has led to two different phages inhibiting RecBCD in two manners.
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Reviewer #2 (Public Review):
This study addresses the ways in which bacteriophages antagonize or coopt the DNA restriction or recombination functions of the bacterial RecBCD helicase-nuclease.
The strength of the paper lies in the marriage of biochemistry and structural biology.
A cryo-EM structure of the RecBCD•gp5.9 complex establishes that gp5.9 is a DNA-mimetic dimer composed of an acidic parallel coiled coil that occupies the dsDNA binding site on the RecB and RecC subunits. The structure of gp5.9 is different from that of the RecBCD-inhibiting DNA mimetic protein phage λ Gam.
Cryo-EM structures of Abc2 are solved in complex with RecBCD bound to a forked DNA duplex, revealing that Abc2 interacts with the RecC subunit. A companion structure is solved containing PPI that copurifies with RecBCD•Abc2.
Whereas the gp5.9 structure fully …
Reviewer #2 (Public Review):
This study addresses the ways in which bacteriophages antagonize or coopt the DNA restriction or recombination functions of the bacterial RecBCD helicase-nuclease.
The strength of the paper lies in the marriage of biochemistry and structural biology.
A cryo-EM structure of the RecBCD•gp5.9 complex establishes that gp5.9 is a DNA-mimetic dimer composed of an acidic parallel coiled coil that occupies the dsDNA binding site on the RecB and RecC subunits. The structure of gp5.9 is different from that of the RecBCD-inhibiting DNA mimetic protein phage λ Gam.
Cryo-EM structures of Abc2 are solved in complex with RecBCD bound to a forked DNA duplex, revealing that Abc2 interacts with the RecC subunit. A companion structure is solved containing PPI that copurifies with RecBCD•Abc2.
Whereas the gp5.9 structure fully rationalizes the effect of gp5.9 on RecBCD activity, the Abc2 structure - while illuminating the docking site on RecBCD, a clear advance - does not clarify how Abc2 impacts RecBCD function.
The authors speculate that Abc2 binding prevents RecA loading on the unwound DNA 3' strand while favoring the loading of the phage recombinase Erf.
Does the structure provide impetus and clues for further experiments to elaborate on that question and, if so, how?
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Reviewer #3 (Public Review):
Wilkinson et al. report the biochemical and structural characterization of two bacteriophage-encoded modifiers of E. coli RecBCD, which has both helicase and nuclease activities. In addition to a function in double-stranded DNA break repair, RecBCD also degrades the genomic DNA of an invading phage and generates phage DNA fragments to be incorporated into CRISPR-based defense systems. Bacteriophages often encode inhibitors to block the RecBCD nuclease activity as the first line of defense. Furthermore, some bacteriophages also encode modifiers of RecBCD to hijack it for phage propagation. The phenomena and effects of phage-encoded Abc2 and Gam were characterized and reported in a series of papers by KC Murphy in the 1990s, of which the 1994 JBC paper is specifically cited as Reference 15.
In this paper, the …
Reviewer #3 (Public Review):
Wilkinson et al. report the biochemical and structural characterization of two bacteriophage-encoded modifiers of E. coli RecBCD, which has both helicase and nuclease activities. In addition to a function in double-stranded DNA break repair, RecBCD also degrades the genomic DNA of an invading phage and generates phage DNA fragments to be incorporated into CRISPR-based defense systems. Bacteriophages often encode inhibitors to block the RecBCD nuclease activity as the first line of defense. Furthermore, some bacteriophages also encode modifiers of RecBCD to hijack it for phage propagation. The phenomena and effects of phage-encoded Abc2 and Gam were characterized and reported in a series of papers by KC Murphy in the 1990s, of which the 1994 JBC paper is specifically cited as Reference 15.
In this paper, the authors chose to study phage T7 encoded RecBCD inhibitor gp5.9 and Salmonella phage P22 encoded RecBCD modifier Abc2. Based on prior knowledge and amino acid composition, it was proposed that gp5.9 is a DNA mimic and blocks DNA binding and hence the enzymatic activity of RecBCD. The authors verified these properties, which are similar to the phage lambda encoded RecBCD inhibitor Gam, whose structure in complex with RecBCD is known. However, gp5.9 shares no sequence similarity with Gam. The cryoEM structure of RecBCD-gp5.9 was thus determined by the authors and reveals that gp5.9 dimerizes to generate a pair of parallel negatively charged alpha helices that mimic a DNA substrate and block DNA binding by RecBCD. Meanwhile, GamS dimerizes in an orthogonal fashion, and only one GamS subunit extends an alpha helix into the DNA binding site of RecBCD. This study shows the diversity in biology and convergent evolution of bacteriophage in blocking RecBCD.
Interestingly, Abc2 cannot be purified by itself alone but is stable only in complexes with RecBCD. Because of a Proline residue (Pro68) in Abc2, which is a substrate of prolyl-isomerase (PPI), WT Abc2 is tightly associated with PPI, but the mutant Abc2P68A can be separated from PPI. Therefore, the authors have prepared both RecBCD- Abc2P68A and RecBCD- Abc2-PPI. The biochemical characterization of the effects of Abc2 on RecBCD is a repeat of KC Murphy's paper, but different from KC Murphy's in the effects of Abc2 on dsDNA-end binding (2-4 fold increase, by Murphy) and helicase activity (3-4 fold reduced, by Murphy) of RecBCD (reference 15). Here, both RecBCD- Abc2P68A and RecBCD- Abc2-PPI have comparable enzymatic activities as RecBCD alone and both can be blocked by gp5.9 as by Gam (Murphy). The cryoEM structures reveal Abc2 binds the Chi-recognition RecC subunit and potentially modifies RecBCD in response to the Chi sequence. But in the absence of DNA, the structure does not explain the in vivo function of Abc2 hijacking RecBCD, nor how Abc2 alters dsDNA binding and helicase activity of RecBCD as reported by Murphy.
The biochemical experiments are expertly carried out. The cryoEM structures are of good quality. While the RecBCD-gp5.9 structure explains the inhibiting mechanism of gp5.9, the lack of functional effects of Abc2 on RecBCD in the in vitro assays is peculiar.
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Author Response:
Reviewer #2 (Public Review):
This study addresses the ways in which bacteriophages antagonize or coopt the DNA restriction or recombination functions of the bacterial RecBCD helicase-nuclease.
The strength of the paper lies in the marriage of biochemistry and structural biology.
A cryo-EM structure of the RecBCD•gp5.9 complex establishes that gp5.9 is a DNA-mimetic dimer composed of an acidic parallel coiled coil that occupies the dsDNA binding site on the RecB and RecC subunits. The structure of gp5.9 is different from that of the RecBCD-inhibiting DNA mimetic protein phage λ Gam.
Cryo-EM structures of Abc2 are solved in complex with RecBCD bound to a forked DNA duplex, revealing that Abc2 interacts with the RecC subunit. A companion structure is solved containing PPI that copurifies with RecBCD•Abc2.
Whereas the …
Author Response:
Reviewer #2 (Public Review):
This study addresses the ways in which bacteriophages antagonize or coopt the DNA restriction or recombination functions of the bacterial RecBCD helicase-nuclease.
The strength of the paper lies in the marriage of biochemistry and structural biology.
A cryo-EM structure of the RecBCD•gp5.9 complex establishes that gp5.9 is a DNA-mimetic dimer composed of an acidic parallel coiled coil that occupies the dsDNA binding site on the RecB and RecC subunits. The structure of gp5.9 is different from that of the RecBCD-inhibiting DNA mimetic protein phage λ Gam.
Cryo-EM structures of Abc2 are solved in complex with RecBCD bound to a forked DNA duplex, revealing that Abc2 interacts with the RecC subunit. A companion structure is solved containing PPI that copurifies with RecBCD•Abc2.
Whereas the gp5.9 structure fully rationalizes the effect of gp5.9 on RecBCD activity, the Abc2 structure - while illuminating the docking site on RecBCD, a clear advance - does not clarify how Abc2 impacts RecBCD function.
The authors speculate that Abc2 binding prevents RecA loading on the unwound DNA 3' strand while favoring the loading of the phage recombinase Erf.
Does the structure provide impetus and clues for further experiments to elaborate on that question and, if so, how?
Regarding the first point (Murphy’s results). We have now included more detail about Murphy’s results and a brief comparative discussion of our own (page 13). An important caveat in interpreting small (<5-fold) effects on RecBCD activity is that the complex is known to possess different levels of specific activity between preparations (from 20% to 100% active based on titration of DNA ends). This is especially problematic when assessing the effect of Abc2 on RecBCD because (unlike gp5.9 for instance) the protein cannot be purified in isolation and titrated into free RecBCD to monitor how activity changes. Instead, one is comparing activity between different preparations either including Abc2 or not. Regarding the second point (how much does the structure tells us about the mechanism of Abc2?). We agree with the general sentiment here: the mechanism of RecBCD hijacking by Abc2 is still a “work in progress”. Nevertheless, the structure is suggestive of effects on Chi recognition and/or RecA loading which is both consistent with biochemical results and suggests new avenues for further investigation.
While the RecBCD-gp5.9 structure “nails” the inhibition mechanism as steric exclusion of substrate, the RecBCD-Abc2 structure is less informative. Previously published biochemical and in vivo analyses of Abc2 show that it modulates rather than completely inhibits the enzyme. The hypothesis is that Abc2 modifies the process of Chi recognition and/or RecA loading (which are themselves coupled processes) in order to facilitate loading of the phage recombinase Erf. Given current structural models for the mechanism of RecBCD, it is not entirely obvious from the structure of RecBCD-Abc2 what exactly this small phage protein is doing, because (a) there is no significant change to the structure of RecBCD induced by Abc2 interaction and (b) no known protein interaction site (eg with RecA) is blocked. Indeed, our original manuscript ended with an acknowledgement that understanding how P22 controls recombination in E. coli was ongoing work. As we see it, in addition to simply revealing the binding site of Abc2, our structure has two significant impacts. Firstly, it is consistent with and extends the existing hypothesis. For example, (a) the interaction of Abc2 with RecC is precisely with the domains of the protein that are responsible for Chi recognition and close to a putative site of RecA loading; (b) the recognition that a conserved proline in Abc2 interacts with the active site of PPI implies that Abc2 function is dependent on proline isomerisation; (c) the possible bridging of RecB and RecC by the C-and N-terminal regions of the protein suggest that Abc2 might hinder intersubunit conformational changes. Secondly, the structure facilitates the testing of this hypothesis. For example, (a) does RecA and/or Erf loading depend on interactions with the surfaces destroyed or created by Abc2 at the interface with RecC (b) does P68A mutation inactivate Abc2?; (c) does failure to recognise and respond to Chi require bridging of RecB and RecC that limits conformational transitions? Crucially, as we explain in the discussion, the future study of the P22 recombination system will require the purification and characterisation of additional factors (Abc2, Arf and Erf) beyond just Abc2. This is something we are working on currently in the lab and consider to be beyond the scope of this work.
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