Structures of RecBCD in complex with phage-encoded inhibitor proteins reveal distinctive strategies for evasion of a bacterial immunity hub.

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.

The type 2 diabetes gene product STARD10 is a phosphoinositide-binding protein that controls insulin secretory granule biogenesis.

OBJECTIVE: Risk alleles for type 2 diabetes at the STARD10 locus are associated with lowered STARD10 expression in the ß-cell, impaired glucose-induced insulin secretion, and decreased circulating proinsulin:insulin ratios. Although likely to serve as a mediator of intracellular lipid transfer, the identity of the transported lipids and thus the pathways through which STARD10 regulates ß-cell function are not understood. The aim of this study was to identify the lipids transported and affected by STARD10 in the ß-cell and the role of the protein in controlling proinsulin processing and insulin granule biogenesis and maturation. METHODS: We used isolated islets from mice deleted selectively in the ß-cell for Stard10 (ßStard10KO) and performed electron microscopy, pulse-chase, RNA sequencing, and lipidomic analyses. Proteomic analysis of STARD10 binding partners was executed in the INS1 (832/13) cell line. X-ray crystallography followed by molecular docking and lipid overlay assay was performed on purified STARD10 protein. RESULTS: ßStard10KO islets had a sharply altered dense core granule appearance, with a dramatic increase in the number of "rod-like" dense cores. Correspondingly, basal secretion of proinsulin was increased versus wild-type islets. The solution of the crystal structure of STARD10 to 2.3 Å resolution revealed a binding pocket capable of accommodating polyphosphoinositides, and STARD10 was shown to bind to inositides phosphorylated at the 3' position. Lipidomic analysis of ßStard10KO islets demonstrated changes in phosphatidylinositol levels, and the inositol lipid kinase PIP4K2C was identified as a STARD10 binding partner. Also consistent with roles for STARD10 in phosphoinositide signalling, the phosphoinositide-binding proteins Pirt and Synaptotagmin 1 were amongst the differentially expressed genes in ßStard10KO islets. CONCLUSION: Our data indicate that STARD10 binds to, and may transport, phosphatidylinositides, influencing membrane lipid composition, insulin granule biosynthesis, and insulin processing.

A conformational switch in response to Chi converts RecBCD from phage destruction to DNA repair.

The RecBCD complex plays key roles in phage DNA degradation, CRISPR array acquisition (adaptation) and host DNA repair. The switch between these roles is regulated by a DNA sequence called Chi. We report cryo-EM structures of the Escherichia coli RecBCD complex bound to several different DNA forks containing a Chi sequence, including one in which Chi is recognized and others in which it is not. The Chi-recognized structure shows conformational changes in regions of the protein that contact Chi and reveals a tortuous path taken by the DNA. Sequence specificity arises from interactions with both the RecC subunit and the sequence itself. These structures provide molecular details for how Chi is recognized and insights into the changes that occur in response to Chi binding that switch RecBCD from bacteriophage destruction and CRISPR spacer acquisition to constructive host DNA repair.

INO80 and SWR1 complexes: the non-identical twins of chromatin remodelling.

The INO80 family of chromatin remodellers are multisubunit complexes that perform a variety of tasks on nucleosomes. Family members are built around a heterohexamer of RuvB-like protein, an ATP-dependent DNA translocase,nuclear actin and actin-related proteins, and a few complex-specific subunits. They modify chromatin in a number of ways including nucleosome sliding and exchange of variant histones. Recent structural information on INO80 and SWR1 complexes has revealed similarities in the basic architecture of the complexes. However, structural and biochemical data on the complexes bound to nucleosomes reveal these similarities to be somewhat superficial and their biochemical activities and mechanisms are very different. Consequently, the INO80 family displays a surprising diversity of function that is based upon a similar structural framework.

Structure of the DNA-Bound Spacer Capture Complex of a Type II CRISPR-Cas System.

CRISPR and associated Cas proteins function as an adaptive immune system in prokaryotes to combat bacteriophage infection. During the immunization step, new spacers are acquired by the CRISPR machinery, but the molecular mechanism of spacer capture remains enigmatic. We show that the Cas9, Cas1, Cas2, and Csn2 proteins of a Streptococcus thermophilus type II-A CRISPR-Cas system form a complex and provide cryoelectron microscopy (cryo-EM) structures of three different assemblies. The predominant form, with the stoichiometry Cas18-Cas24-Csn28, referred to as monomer, contains ∼30 bp duplex DNA bound along a central channel. A minor species, termed a dimer, comprises two monomers that sandwich a further eight Cas1 and four Cas2 subunits and contains two DNA ∼30-bp duplexes within the channel. A filamentous form also comprises Cas18-Cas24-Csn28 units (typically 2-6) but with a different Cas1-Cas2 interface between them and a continuous DNA duplex running along a central channel.

CtIP forms a tetrameric dumbbell-shaped particle which bridges complex DNA end structures for double-strand break repair.

CtIP is involved in the resection of broken DNA during the S and G2 phases of the cell cycle for repair by recombination. Acting with the MRN complex, it plays a particularly important role in handling complex DNA end structures by localised nucleolytic processing of DNA termini in preparation for longer range resection. Here we show that human CtIP is a tetrameric protein adopting a dumbbell architecture in which DNA binding domains are connected by long coiled-coils. The protein complex binds two short DNA duplexes with high affinity and bridges DNA molecules in trans. DNA binding is potentiated by dephosphorylation and is not specific for DNA end structures per se. However, the affinity for linear DNA molecules is increased if the DNA terminates with complex structures including forked ssDNA overhangs and nucleoprotein conjugates. This work provides a biochemical and structural basis for the function of CtIP at complex DNA breaks.

DNA translocation mechanism of an XPD family helicase.

The XPD family of helicases, that includes human disease-related FANCJ, DDX11 and RTEL1, are Superfamily two helicases that contain an iron-sulphur cluster domain, translocate on ssDNA in a 5'-3' direction and play important roles in genome stability. Consequently, mutations in several of these family members in eukaryotes cause human diseases. Family members in bacteria, such as the DinG helicase from Escherichia coli, are also involved in DNA repair. Here we present crystal structures of complexes of DinG bound to single-stranded DNA (ssDNA) in the presence and absence of an ATP analogue (ADP•BeF3), that suggest a mechanism for 5'-3' translocation along the ssDNA substrate. This proposed mechanism has implications for how those enzymes of the XPD family that recognise bulky DNA lesions might stall at these as the first step in initiating DNA repair. Biochemical data reveal roles for conserved residues that are mutated in human diseases.

Structure and dynamics of the yeast SWR1-nucleosome complex.

The yeast SWR1 complex exchanges histone H2A in nucleosomes with Htz1 (H2A.Z in humans). The cryo-electron microscopy structure of the SWR1 complex bound to a nucleosome at 3.6-angstrom resolution reveals details of the intricate interactions between components of the SWR1 complex and its nucleosome substrate. Interactions between the Swr1 motor domains and the DNA wrap at superhelical location 2 distort the DNA, causing a bulge with concomitant translocation of the DNA by one base pair, coupled to conformational changes of the histone core. Furthermore, partial unwrapping of the DNA from the histone core takes place upon binding of nucleosomes to SWR1 complex. The unwrapping, as monitored by single-molecule data, is stabilized and has its dynamics altered by adenosine triphosphate binding but does not require hydrolysis.

Structure and regulation of the human INO80-nucleosome complex.

Access to DNA within nucleosomes is required for a variety of processes in cells including transcription, replication and repair. Consequently, cells encode multiple systems that remodel nucleosomes. These complexes can be simple, involving one or a few protein subunits, or more complicated multi-subunit machines 1 . Biochemical studies2-4 have placed the motor domains of several chromatin remodellers in the superhelical location 2 region of the nucleosome. Structural studies of yeast Chd1 and Snf2-a subunit in the complex with the capacity to remodel the structure of chromatin (RSC)-in complex with nucleosomes5-7 have provided insights into the basic mechanism of nucleosome sliding performed by these complexes. However, how larger, multi-subunit remodelling complexes such as INO80 interact with nucleosomes and how remodellers carry out functions such as nucleosome sliding 8 , histone exchange 9 and nucleosome spacing10-12 remain poorly understood. Although some remodellers work as monomers 13 , others work as highly cooperative dimers11, 14, 15. Here we present the structure of the human INO80 chromatin remodeller with a bound nucleosome, which reveals that INO80 interacts with nucleosomes in a previously undescribed manner: the motor domains are located on the DNA at the entry point to the nucleosome, rather than at superhelical location 2. The ARP5-IES6 module of INO80 makes additional contacts on the opposite side of the nucleosome. This arrangement enables the histone H3 tails of the nucleosome to have a role in the regulation of the activities of the INO80 motor domain-unlike in other characterized remodellers, for which H4 tails have been shown to regulate the motor domains.

Cryo-EM structures of the human INO80 chromatin-remodeling complex.

Access to chromatin for processes such as transcription and DNA repair requires the sliding of nucleosomes along DNA. This process is aided by chromatin-remodeling complexes, such as the multisubunit INO80 chromatin-remodeling complex. Here we present cryo-EM structures of the active core complex of human INO80 at 9.6 Å, with portions at 4.1-Å resolution, and reconstructions of combinations of subunits. Together, these structures reveal the architecture of the INO80 complex, including Ino80 and actin-related proteins, which is assembled around a single RUVBL1 (Tip49a) and RUVBL2 (Tip49b) AAA+ heterohexamer. An unusual spoked-wheel structural domain of the Ino80 subunit is engulfed by this heterohexamer; both, in combination, form the core of the complex. We also identify a cleft in RUVBL1 and RUVBL2, which forms a major interaction site for partner proteins and probably communicates these interactions to its nucleotide-binding sites.

Production and Assay of Recombinant Multisubunit Chromatin Remodeling Complexes.

We have developed a novel system to facilitate the rapid and easy cloning of multiple genes (>10) in under a week. Using this system we have been able to successfully clone, overexpress, and purify a number of large multimeric proteins from insect cells, including the chromatin remodeling complexes SWR1 and INO80. Using Förster resonance energy transfer (FRET)-based assays we have demonstrated that our overexpressed enzymes have activities comparable to those purified from sources where the proteins are expressed under their endogenous promoters.

Crosstalk within a functional INO80 complex dimer regulates nucleosome sliding.

Several chromatin remodellers have the ability to space nucleosomes on DNA. For ISWI remodellers, this involves an interplay between H4 histone tails, the AutoN and NegC motifs of the motor domains that together regulate ATPase activity and sense the length of DNA flanking the nucleosome. By contrast, the INO80 complex also spaces nucleosomes but is not regulated by H4 tails and lacks the AutoN and NegC motifs. Instead nucleosome sliding requires cooperativity between two INO80 complexes that monitor DNA length simultaneously on either side of the nucleosome during sliding. The C-terminal domain of the human Ino80 subunit (Ino80CTD) binds cooperatively to DNA and dimerisation of these domains provides crosstalk between complexes. ATPase activity, rather than being regulated, instead gradually becomes uncoupled as nucleosome sliding reaches an end point and this is controlled by the Ino80CTD. A single active ATPase motor within the dimer is sufficient for sliding.

Functional characterization and architecture of recombinant yeast SWR1 histone exchange complex.

We have prepared recombinant fourteen subunit yeast SWR1 complex from insect cells using a modified MultiBac system. The 1.07 MDa recombinant protein complex has histone-exchange activity. Full exchange activity is realized with a single SWR1 complex bound to a nucleosome. We also prepared mutant complexes that lack a variety of subunits or combinations of subunits and these start to reveal roles for some of these subunits as well as indicating interactions between them in the full complex. Complexes containing a series of N-terminally and C-terminally truncated Swr1 subunits reveal further details about interactions between subunits as well as their binding sites on the Swr1 subunit. Finally, we present electron microscopy studies revealing the dynamic nature of the complex and a 21 Å resolution reconstruction of the intact complex provides details not apparent in previously reported structures, including a large central cavity of sufficient size to accommodate a nucleosome.

The structure and function of an RNA polymerase interaction domain in the PcrA/UvrD helicase.

The PcrA/UvrD helicase functions in multiple pathways that promote bacterial genome stability including the suppression of conflicts between replication and transcription and facilitating the repair of transcribed DNA. The reported ability of PcrA/UvrD to bind and backtrack RNA polymerase (1,2) might be relevant to these functions, but the structural basis for this activity is poorly understood. In this work, we define a minimal RNA polymerase interaction domain in PcrA, and report its crystal structure at 1.5 Å resolution. The domain adopts a Tudor-like fold that is similar to other RNA polymerase interaction domains, including that of the prototype transcription-repair coupling factor Mfd. Removal or mutation of the interaction domain reduces the ability of PcrA/UvrD to interact with and to remodel RNA polymerase complexes in vitro. The implications of this work for our understanding of the role of PcrA/UvrD at the interface of DNA replication, transcription and repair are discussed.

Structural basis for the inhibition of RecBCD by Gam and its synergistic antibacterial effect with quinolones.

Our previous paper (Wilkinson et al, 2016) used high-resolution cryo-electron microscopy to solve the structure of the Escherichia coli RecBCD complex, which acts in both the repair of double-stranded DNA breaks and the degradation of bacteriophage DNA. To counteract the latter activity, bacteriophage λ encodes a small protein inhibitor called Gam that binds to RecBCD and inactivates the complex. Here, we show that Gam inhibits RecBCD by competing at the DNA-binding site. The interaction surface is extensive and involves molecular mimicry of the DNA substrate. We also show that expression of Gam in E. coli or Klebsiella pneumoniae increases sensitivity to fluoroquinolones; antibacterials that kill cells by inhibiting topoisomerases and inducing double-stranded DNA breaks. Furthermore, fluoroquinolone-resistance in K. pneumoniae clinical isolates is reversed by expression of Gam. Together, our data explain the synthetic lethality observed between topoisomerase-induced DNA breaks and the RecBCD gene products, suggesting a new co-antibacterial strategy.

Mechanism for nuclease regulation in RecBCD.

In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is catalysed by AddAB, AdnAB or RecBCD-type helicase-nucleases. These enzyme complexes are highly processive, duplex unwinding and degrading machines that require tight regulation. Here, we report the structure of E.coli RecBCD, determined by cryoEM at 3.8 Å resolution, with a DNA substrate that reveals how the nuclease activity of the complex is activated once unwinding progresses. Extension of the 5'-tail of the unwound duplex induces a large conformational change in the RecD subunit, that is transferred through the RecC subunit to activate the nuclease domain of the RecB subunit. The process involves a SH3 domain that binds to a region of the RecB subunit in a binding mode that is distinct from others observed previously in SH3 domains and, to our knowledge, this is the first example of peptide-binding of an SH3 domain in a bacterial system.

Synergy and antagonism in regulation of recombinant human INO80 chromatin remodeling complex.

We have purified a minimal core human Ino80 complex from recombinant protein expressed in insect cells. The complex comprises one subunit each of an N-terminally truncated Ino80, actin, Arp4, Arp5, Arp8, Ies2 and Ies6, together with a single heterohexamer of the Tip49a and Tip49b proteins. This core complex has nucleosome sliding activity that is similar to that of endogenous human and yeast Ino80 complexes and is also inhibited by inositol hexaphosphate (IP6). We show that IP6 is a non-competitive inhibitor that acts by blocking the stimulatory effect of nucleosomes on the ATPase activity. The IP6 binding site is located within the C-terminal region of the Ino80 subunit. We have also prepared complexes lacking combinations of Ies2 and Arp5/Ies6 subunits that reveal regulation imposed by each of them individually and synergistically that couples ATP hydrolysis to nucleosome sliding. This coupling between Ies2 and Arp5/Ies6 can be overcome in a bypass mutation of the Arp5 subunit that is active in the absence of Ies2. These studies reveal several underlying mechanisms for regulation of ATPase activity involving a complex interplay between these protein subunits and IP6 that in turn controls nucleosome sliding.

Chi hotspots trigger a conformational change in the helicase-like domain of AddAB to activate homologous recombination.

In bacteria, the repair of double-stranded DNA breaks is modulated by Chi sequences. These are recognised by helicase-nuclease complexes that process DNA ends for homologous recombination. Chi activates recombination by changing the biochemical properties of the helicase-nuclease, transforming it from a destructive exonuclease into a recombination-promoting repair enzyme. This transition is thought to be controlled by the Chi-dependent opening of a molecular latch, which enables part of the DNA substrate to evade degradation beyond Chi. Here, we show that disruption of the latch improves Chi recognition efficiency and stabilizes the interaction of AddAB with Chi, even in mutants that are impaired for Chi binding. Chi recognition elicits a structural change in AddAB that maps to a region of AddB which resembles a helicase domain, and which harbours both the Chi recognition locus and the latch. Mutation of the latch potentiates the change and moderately reduces the duration of a translocation pause at Chi. However, this mutant displays properties of Chi-modified AddAB even in the complete absence of bona fide hotspot sequences. The results are used to develop a model for AddAB regulation in which allosteric communication between Chi binding and latch opening ensures quality control during recombination hotspot recognition.

Structural features of Chi recognition in AddAB with implications for RecBCD.

AddAB and RecBCD-type helicase-nuclease complexes control the first stage of bacterial homologous recombination (HR) - the resection of double strand DNA breaks. A switch in the activities of the complexes to initiate repair by HR is regulated by a short, species-specific DNA sequence known as a Crossover Hotspot Instigator (Chi) site. It has been shown that, upon encountering Chi, AddAB and RecBCD pause translocation before resuming at a reduced rate. Recently, the structure of B.subtilis AddAB in complex with its regulatory Chi sequence revealed the nature of Chi binding and the paused translocation state. Here the structural features associated with Chi binding are described in greater detail and discussed in relation to the related E.coli RecBCD system.