The processing of double-stranded DNA breaks for recombinational repair by helicase–nuclease complexes

DNA Repair 9 (2010) 276–285

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Mini-review

The processing of double-stranded DNA breaks for recombinational repair by helicase–nuclease complexes Joseph T.P. Yeeles, Mark S. Dillingham ∗ DNA-Protein Interactions Unit, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, United Kingdom

a r t i c l e

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Article history: Available online 29 January 2010 Keywords: AddAB RecBCD Dna2 Helicase–nuclease Homologous recombination Double-stranded DNA break repair

a b s t r a c t Double-stranded DNA breaks are prepared for recombinational repair by nucleolytic digestion to form single-stranded DNA overhangs that are substrates for RecA/Rad51-mediated strand exchange. This processing can be achieved through the activities of multiple helicases and nucleases. In bacteria, the function is mainly provided by a stable multi-protein complex of which there are two structural classes; AddABand RecBCD-type enzymes. These helicase–nucleases are of special interest with respect to DNA helicase mechanism because they are exceptionally powerful DNA translocation motors, and because they serve as model systems for both single molecule studies and for understanding how DNA helicases can be coupled to other protein machinery. This review discusses recent developments in our understanding of the AddAB and RecBCD complexes, focussing on their distinctive strategies for processing DNA ends. We also discuss the extent to which bacterial DNA end resection mechanisms may parallel those used in eukaryotic cells. © 2010 Elsevier B.V. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Strategies for the repair of double-stranded DNA breaks in bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Initiation of double-stranded DNA break repair by helicase–nucleases and the recombination hotspot Chi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Distinctive structural classes of helicase–nuclease complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanism of RecBCD-type helicase–nucleases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Dual motor–single nuclease mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Control of RecBCD by Chi sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. DNA translocation properties of RecBCD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. RecA loading mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanism of AddAB-type helicase–nucleases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Single motor–dual nuclease mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Models for the interaction of AddAB with Chi sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. An essential iron–sulphur cluster in B. subtilis AddAB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Comparison between bacterial helicase–nuclease mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Emerging parallels between bacterial and eukaryotic DNA end resection? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction 1.1. Strategies for the repair of double-stranded DNA breaks in bacteria

∗ Corresponding author. Tel.: +44 0117 3312159; fax: +44 0117 3312168. E-mail address: [email protected] (M.S. Dillingham). 1568-7864/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2009.12.016

In bacteria, as in eukaryotes, double-stranded DNA breaks (DSBs) can be produced by a variety of different mechanisms [1]. They may be formed directly by ionising radiation, DNA damaging

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Fig. 1. The recombination-dependent repair of collapsed replication forks. The recombination-dependent replication pathway is illustrated, together with the protein components from both E. coli and B. subtilis. Proteins that have been implicated in the repair pathway but whose functions remain unclear are indicated with a question mark. (A) Replication forks that run into nicks in the leading strand template will collapse, generating an intact chromosome and a detached chromosome arm. Detached chromosome arms can also be formed by cleavage of replication forks that have undergone reversal due to replisome stalling [3,6]. (B) The detached arm is principally targeted by helicase–nuclease complexes, which resect the DNA end to form a 3 -ssDNA overhang in a Chi-dependent reaction. An alternative DNA end resection pathway is thought to operate in wild type B. subtilis cells [110,132] and RecBCD E. coli cells carrying additional suppressor mutations [4]. The pathway uses the combined activities of the RecQ helicase and the RecJ exonuclease. Genetic studies have also implicated several additional proteins in the initial stages of DNA end resection, but the precise functions of these proteins during double-strand break repair remains to be elucidated [9]. (C) The RecA protein is loaded onto the 3 -ssDNA overhang, and the resulting RecA filament catalyses the re-attachment of the broken arm with a homologous region of the sister chromosome. (D) The resulting D-loop structure is subsequently targeted by the replication-restart protein PriA. (D and E) PriA-dependent replisome assembly and Holliday junction resolution then gives rise to a fully-functional replisome [13].

agents, desiccation, mechanical stress or unregulated nuclease activities. Alternatively, a major source of DSBs occurs indirectly through the interplay of DNA replication with damaged DNA or other replication fork barriers (Fig. 1) [2–7]. DSBs are potentially lethal and so bacterial cells have evolved a range of machinery to repair them [8–10]. There are two fundamentally different approaches for the repair of the breaks, non-homologous end joining (NHEJ) and homologous recombination (HR). Repair by NHEJ, which is only present in a subset of bacteria, involves the bridging and ligation of the two free ends of a DSB [11]. This error-prone process is probably only used when a homologous donor DNA is not readily available, for example during prolonged stationary phase in specialised cell-cycle stages such as sporulation [10,12]. In contrast the HR mechanism is essentially ubiquitous, more prominent and error-free. This process relies on the presence of a homologous donor DNA (generally the sister chromatid) to act as a template

for faithful repair [5]. The pathway for DSB repair by HR may be thought of as occurring in three stages. In the pre-synaptic step, the DNA ends are processed to form single-stranded DNA overhangs that are suitable substrates for the binding of RecA protein. During synapsis, the RecA nucleoprotein filament performs a homology search and catalyses strand exchange with a donor DNA molecule to form a joint molecule. This leads to the establishment of Holliday junctions and allows the copying of information from the donor duplex. The final post-synaptic step involves resolution of the junctions to form repaired products. In recent years, it has become increasingly evident that another key role for recombinational repair is to salvage the single end breaks that are formed when replication forks collapse (reviewed in Ref. [5]). Much of the same HR machinery, along with specialised replication-restart proteins [13], can re-prime DNA replication from a DNA break in an origin-independent manner ([5]; Fig. 1).

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1.2. Initiation of double-stranded DNA break repair by helicase–nucleases and the recombination hotspot Chi In both the DSB repair and recombination-dependent replication pathways, the broken DNA ends are first processed by a multi-protein machine containing helicase and nuclease activities, which we will refer to generically as a helicase–nuclease complex [4]. Such complexes can be sub-divided into two major structural classes, exemplified by the B. subtilis AddAB and Escherichia coli RecBCD proteins, and referred to hereafter as AddAB-type or RecBCD-type enzymes [14]. Regardless of the structural class, the function is the same: the helicase–nuclease directs free DNA ends into the pathway for HR by converting them into RecA nucleoprotein filaments in a manner that is regulated by the recombination hotspot sequence Chi (crossover hotspot instigator) ([1,14]; Fig. 2). The complex binds with high affinity to blunt or nearly-blunt DNA ends [15–17]. Using the free energy derived from ATP hydrolysis, it then tracks into the duplex and concomitantly separates it into the component single-strands, which we will refer to as the 3 > 5 and 5 > 3 strands (Fig. 2). The nascent ssDNA is rapidly bound and stabilised by the non-specific single-stranded DNA binding protein (SSB). During movement along the duplex, both singlestrands of DNA are intermittently cleaved. The phosphodiester bond hydrolysis is apparently stochastic, such that the density of cleavage positions in the single-strand products is dependent on both the speed of translocation and the rate of the nuclease activity [1,18]. This behaviour continues until the translocating enzyme encounters the regulatory sequence Chi. Following Chi recognition, processive helicase activity (i.e. DNA translocation and unwinding)

is maintained, but a final cleavage is made on the 3 > 5 strand and further cleavage beyond Chi on the same strand is essentially eliminated [19–21]. Cleavage of the 5 > 3 strand continues and, consequently, the net product is a long, 3 -ssDNA overhang terminating very close to the Chi sequence. This is subsequently coated with RecA protein to form an active nucleoprotein filament that facilitates the subsequent steps of recombinational repair (Fig. 1). However, because SSB binds to ssDNA extremely rapidly and tightly, RecA is generally unable to effectively compete for binding to the ssDNA products. This problem is circumvented by actively loading the RecA protein onto the Chi-terminated ssDNA overhang [22]. This end processing reaction, including the downstream coupling to strand exchange with a homologous donor and even replication-restart, can be fully recapitulated in vitro using model substrates and purified proteins [23,24]. A remarkable feature of the activity of helicase–nuclease complexes is their control by the regulatory sequence Chi (for an interesting historical perspective on the discovery of Chi see [25]). Recognition of Chi has many effects on the helicase–nuclease complex (Sections 2.2 and 3.2) which, together, switch the enzyme from a destructive degradative mode of action to a recombination-promoting mode. Chi sequences have been discovered and characterised in several bacteria [26–32]. They are significantly over-represented, being especially concentrated in core regions of the genome that are conserved between different strains of the same species [31]. The Chi sequence is asymmetric and recognised in only one orientation. Importantly, the Chi sequences of bacterial genomes are skewed such that most face towards the origin of replication; the appropriate orientation for promoting recombination from collapsed replication forks. This observation lends weight to the idea that the major function of the helicase–nuclease complex is in the support of interrupted DNA replication (Fig. 1; [2]). Note that the lengths and exact sequences of Chi vary between different bacterial species and their resident helicase–nuclease. This is true even for members of the same class of enzyme. For example, E. coli RecBCD recognises the octameric sequence 5 -GCTGGTGG-3 [33], whereas L. lactis RexAB (an AddAB-type enzyme) recognises the heptameric sequence 5 -GCGCGTG-3 , and B. subtilis AddAB the pentameric sequence 5 -AGCGG-3 [27]. Nevertheless, there is some indication that Chi is conserved between closely-related bacterial species [31,32]. All known Chi sequences are rich in guanine residues and may be embedded in GT-rich regions of sequence that are preferred substrates for RecA-mediated strand exchange [34]. 1.3. Distinctive structural classes of helicase–nuclease complex

Fig. 2. DNA end processing catalysed by helicase–nuclease complexes. (1) Helicase–nuclease enzymes (grey and yellow rectangles) bind tightly to blunt (or nearly-blunt) duplex DNA ends. (2) They use ATP-dependent motors to unwind the dsDNA into its component single-strands, whilst concomitantly degrading both the nascent 3 > 5 (black) and 5 > 3 (pink) strands. (3) DNA unwinding and the degradation of both single-strands persists until the regulatory sequence Chi is recognised. (4) Following Chi recognition, the enzymes continue to unwind the duplex and cleave the 5 > 3 strand, whilst nucleolytic cleavage of the 3 > 5 strand is attenuated. This Chi-dependent modulation of activity results in the formation of a 3 -terminated ssDNA tail. (5) The E. coli RecBCD enzyme facilitates the loading of RecA (purple ovals) onto the 3 > 5 strand in the presence of SSB. At present, it is unknown whether or not the ability to load RecA is a universal feature of helicase–nuclease complexes (Section 3.2).

The vast majority of sequenced bacterial species encode a single AddAB- or RecBCD-type helicase–nuclease (Fig. 3), and the genes for the component proteins are usually arranged in an operon structure [35]. The AddAB enzymes are composed of two subunits (AddA and AddB), whereas RecBCD is a heterotrimer of three polypeptides (RecB, RecC and RecD) (Fig. 4). There are occasional examples of bacteria which contain multiple complexes of the same type, both classes of enzyme, or neither (for a more complete discussion see [35]). AddAB-type complexes were originally thought to be restricted to a fairly limited niche in the Bacillus/Clostridium subdivision of Gram-positive bacteria [14], but are now appreciated to be relatively abundant [35–37] and are in fact marginally more common than the RecBCD complexes. Structural homologues of the AddAB and RecBCD complexes are not found in eukaryotes, although there are several helicases and nucleases implicated in the resection of DNA ends and we will return to this point later in the review. AddAB complexes are occasionally found in Archaea although their function, if any, in these organisms remains to be determined [35]. Although the AddAB- and RecBCD-type

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Fig. 3. Phylogenetic distribution of AddAB- and RecBCD-type helicase–nucleases. The schematic tree is based on 16S ribosomal RNA sequence comparisons. Taxa where helicase–nuclease complexes have not been identified are coloured black. Groups of bacteria containing solely AddAB or solely RecBCD are coloured green and blue respectively. Groups coloured red contain bacteria that possess either AddAB or RecBCD, but not both enzymes within the same bacterial species. (*) Several members of the Actinobacteria contain putative AddAB- and RecBCD-type enzymes in the same organism. However, the biological function of these enzymes is yet to be determined. (**) Members of the Thermus/Deinococcus phylum contain intact AddA genes adjacent to AddB-like genes that lack the nuclease domain. (The figure is modified from Ref. [14] but has been substantially updated to include more recent data [35,36])

complexes are structurally distinct, they are functionally analogous. Bacteria deficient in either AddAB or RecBCD activity display reduced viability and defective DSB repair, but are also compromised in other processes involving recombination initiated at free DNA ends, for example conjugal and transductional recombination ([1,14] and references therein). Compelling evidence that AddAB and RecBCD complexes play equivalent roles in cells is provided by the observation that AddAB-type complexes can be successfully substituted for RecBCD in vivo [28,38–41]. E. coli RecBCD was also shown to be involved in the recombinational repair of DNA that is cross-linked to proteins [42]. Furthermore, RecBCD promotes E. coli viability in a manner that is independent of recombination, which

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probably reflects its ability to degrade toxic intermediates of replication or to reset reversed replication forks [40,43]. Interestingly, the expression of AddAB was shown to be linked to the development of competence in B. subtilis and AddAB is required for the recombination of incoming DNA [44]. A theme is currently emerging that helicase–nuclease activity protects some pathogenic bacteria against harsh environments encountered within the host. For example, AddAB from Coxiella burnetii (the causative agent of Q-fever) is up-regulated under conditions of oxidative stress. This probably helps to protect this bacterium against the high concentration of reactive oxygen species found in the lysosome-like vacuole within which it colonises the host cell [41]. Helicobacter pylori AddAB was shown to be required for efficient colonisation of the mouse stomach [45]. It promotes the recombination-dependent variation of cell surface receptors that helps the bacterium to evade the adaptive immune system of the host. Moreover, its capacity to promote DSB repair probably helps to reduce the killing power of oxidative attack from host macrophages. Similarly, the viability and virulence of E. coli, Neisseria gonorrhoeae and Salmonella may be reduced by loss of RecBCD function because the bacteria are sensitive to the damaging effects of reactive oxygen species and nitric oxide (both excreted at high concentrations by macrophages) or bile salts [46–50]. Consequently, helicase–nucleases are, in principle, a possible target for therapeutics against bacteria that cause human disease. 2. Mechanism of RecBCD-type helicase–nucleases The prototypical RecBCD-type enzyme from E. coli has been the subject of study for nearly half a century. Since its discovery as a powerful ATP-dependent exonuclease activity [51–54] a large body of genetic, biochemical and structural work from many laboratories has resulted in a fairly sophisticated understanding of the function of this complex (reviewed in Ref. [1]). 2.1. Dual motor–single nuclease mechanism RecBCD is a 330 kDa heterotrimer composed of the RecB, RecC and RecD polypeptides (Fig. 4 and Fig. 5). RecB and RecD both contain a Superfamily 1 helicase domain: a device that couples ATP hydrolysis to unidirectional translocation on single-stranded DNA [55] and which is found in many DNA repair enzymes including E. coli UvrD and S. cerevisiae Srs2. The catalytic core of SF1 helicases consists of two neighbouring domains, each with a RecA-like fold, that bind ATP in an intervening cleft. Single-stranded DNA binds

Fig. 4. Primary structure and domain architecture of AddAB and RecBCD complexes. E. coli RecBCD is a heterotrimeric complex. The N-terminal region of the RecB polypeptide contains the seven motifs that are characteristic of a SF1A helicase domain, whilst the C-terminal region harbours three Superfamily 1 nuclease motifs, in addition to a motif specific to RecB-family nuclease domains (F). The RecC subunit does not possess any conserved motifs. However, the N-terminal region of the protein shares structural homology with SF1 helicases and this region has been implicated in Chi recognition, whilst the C-terminal region contains an inactive nuclease-like domain. The RecD polypeptide contains seven conserved SF1B helicase motifs. B. subtilis AddAB consists of only two subunits. AddA is homologous to RecB and contains an N-terminal SF1A helicase domain and a C-terminal RecB-family nuclease domain. The N-terminal region of AddB contains an intact Walker-A motif (equivalent to helicase motif 1), but apparently lacks the remaining helicase motifs. This region also shares weak sequence identity with SF1A helicases and the RecC protein suggesting that, like RecC, AddB shares structural homology with SF1 helicases. Additionally, AddB contains an iron-staple nuclease domain at its C-terminus (Section 3.3). The positions of four cysteine residues responsible for Fe–S cluster co-ordination are indicated (C) with the number of cysteine residues present at each location indicated in superscript.

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Fig. 5. Structure and proposed mechanism for the RecBCD-type helicase–nuclease. (A) Surface representation of the RecBCD crystal structure that has been cut back to reveal the ssDNA tails and the channels (yellow) through which the single-strands are proposed to run. The domains of the complex are coloured according to the primary structure diagram in Fig. 4, with solid colours representing active domains and striped regions denoting inactivated domains. (B) Cartoon illustration of the RecBCD complex prior to Chi recognition. The domains are colour-coded as above. Arrows and circles represent helicase and nuclease domains respectively, and the arrow direction indicates the direction of helicase movement relative to ssDNA. The dual motor organisation (red and green arrows) delivers both single-strands to the nuclease domain (blue circle) at the rear of the enzyme. (C) Chi recognition is manifest as the Chi-scanning region of RecC (striped red arrow) binding to Chi, thus preventing the 3 > 5 strand from accessing the nuclease domain. This sequestration of the Chi-containing strand is predicted to result in a growing ssDNA loop in between the RecB helicase domain (red arrow) and RecC Chi-scanning domain (striped red arrow).

across the top surface of both domains trapping several nucleobases in pockets on the protein surface. The repeated binding and hydrolysis of ATP in between the two domains leads to cyclic conformational changes that pass the nucleobases across the binding pockets on the top surface [56,57]. There are two mechanistic variants of SFI enzymes, SF1A and SF1B, which pass the singlestranded DNA across the RecA-like domains in the 3 > 5 and 5 > 3 directions respectively [55]. RecBCD contains one motor of each polarity: RecB is a SF1A helicase [58], whereas RecD is a member of SF1B [59]. Together, the two helicases drive movement of the RecBCD complex along the DNA using a bipolar translocation mechanism in which each motor engages with one single-strand of DNA [59,60]. The motors can function autonomously and move at unequal speeds. RecD seems to act as the lead helicase in the wild type complex [60,61], but the factors affecting the relative speed of the two motors are complex and may be condition-dependent (see [1] and references therein for discussion). As the enzyme unwinds DNA, the nascent single-strands are cut asymmetrically with the 3 > 5 strand being degraded more vigorously than the 5 > 3 strand [21]. This activity maps to the C-terminus of RecB, which contains the prototypical member of the “RecB-family” of nuclease domains [62]. This domain alone is responsible for all cleavages made on both strands of the DNA [63,64]. Therefore, the RecBCDtype complexes use a “dual motor–single nuclease” mechanism for processive DNA end resection (Fig. 5). 2.2. Control of RecBCD by Chi sequences Following Chi recognition on the 3 > 5 strand, the cleavage of that strand ceases, but cleavage of the 5 > 3 strand is actually upregulated [19,21]. Although reductionist experiments on isolated component proteins (and domains therein) had established the location and number of helicase and nuclease active sites, they had largely failed to address how these activities were controlled by Chi to produce the recombinogenic 3 -ssDNA tail. By revealing the relative positioning of these domains within the whole complex, the crystal structure of RecBCD [65] provided significant insight into the regulation of nuclease activity by recombination hotspots. The salient observations from the structure (Fig. 5) and other subse-

quent analyses [66,67] are as follows. First, the N- and C-terminal domains of RecC are structurally related to a SF1 DNA helicase and a nuclease domain respectively, but the complete lack of the key catalytic residues suggests that both of these domains have been “inactivated”. Second, the nascent single-strands of DNA pass through two long channels in the complex (Fig. 5A, yellow surface) where they encounter the various functional domains in a specific order and converge on the nuclease domain at the rear of the enzyme. Third, the SF1A motor and the inactivated helicase domain lie on the path of the 3 > 5 strand whereas the inactivated nuclease-like domain and the SF1B motor lie on the path of the 5 > 3 strand. Finally, with respect to the direction of movement of the complex, the inactivated helicase domain of RecC forms part of the 3 > 5 channel immediately after the SF1A motor but before the nuclease domain. The three protein subunits are intimately woven together, coupling their activities in a manner that suggests a simple mechanism for the regulation of DNA end resection by Chi (Fig. 5B and C). Before Chi recognition, both strands pass completely through the complex and are cut by the nuclease domain as they emerge at the rear (Fig. 5B). Importantly, the inactivated helicase domain is positioned such that, if it were to search for a Chi sequence during translocation, then its binding to Chi would prevent further hydrolysis of the 3 > 5 strand (Fig. 5C). The idea that the RecC inactivated helicase domain acts as a “scanner” for the Chi sequence makes good sense for several reasons [1,65]. Most persuasively, residues known to be involved in the specificity of Chi recognition [68,69] map precisely to the channel in the RecC helicase-like domains (Fig. 5A). The “Chiscanning” model further predicts that the SF1A motor is required to deliver the Chi sequence to the scanning channel, whereas the SF1B motor should be dispensable, and this was recently confirmed experimentally [70]. Because DNA translocation and unwinding continue following Chi recognition, the model further implies that a loop of ssDNA (part of the 3 > 5 strand) should be extruded from the complex after hotspot recognition. This putative loop is an important recombination intermediate, since it is destined to bind the RecA protein and participate in strand exchange with the homologous donor duplex, but its existence has never been directly demonstrated. Some evidence for this loop was provided

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by the observation of a “fluorescent ellipsoid” of labelled DNA travelling with RecBCD following Chi recognition in a single molecule study of DNA unwinding [71]. Furthermore, biochemical and in silico analysis of the binding of RecBCD to DNA ends containing synthetic pre-formed overhangs identified the formation of ssDNA loops in the 3 -tail, which may be accommodated between the RecB and RecC subunits, mimicking the structures formed after Chi recognition [17,72]. Exactly how the growing ssDNA loop might be released to allow plectonemic strand exchange, or how the translocating RecBCD enzyme escapes from the processed DNA molecule remain unclear. Confirmation of the “Chi-scanning” hypothesis will require further work including site-directed mutagenesis of the proposed recognition channel or, ultimately, structural information for a RecBCD-Chi recognition complex. This challenging goal might prove to be more tractable using the AddAB-type enzymes (Section 3.2). However, it is anticipated that experiments of this type will provide important new information on the structural basis for sequence-specific ssDNA recognition and further insight into how the “helicase” domain has been modified to accomplish such a task. 2.3. DNA translocation properties of RecBCD The DNA translocation properties of RecBCD have attracted special attention in the DNA helicase community because RecBCD is the fastest (∼1–2 kbp/s) and most processive (∼30 kbp) DNA helicase characterised to date [71,73–76]. These properties are at least partially explained by the unusual use of two DNA motors to drive translocation [77], and have made RecBCD particularly amenable to study using single molecule techniques. These experiments have yielded many novel insights into the translocation mechanism including population heterogeneity in the rate [73] as well as rate changes, stalling and back-sliding under a restraining force [78]. Another study identified a long pause at Chi followed by a reduction in the translocation rate post-Chi [71]. It was suggested that this behaviour might assist RecBCD in cleaving the 3 -ssDNA at Chi and in loading the RecA protein. It was subsequently shown that the rate reduction after Chi was associated with a switch in the lead motor subunit from RecD to RecB [61]. Direct observation of RecBCD movement along DNA also revealed that the heterotrimer remained intact following Chi recognition, which disproved the long-standing hypothesis that ejection of RecD was part of the mechanism for the response to Chi [79,80]. 2.4. RecA loading mechanism The final task of RecBCD is to load RecA protein onto the Chiterminated single-strand [22]. The purified RecB nuclease domain binds to RecA in vitro [81]. Based on that observation, a model for RecA loading was proposed whereby Chi recognition unmasked a cryptic RecA binding locus on the nuclease domain that was equivalent to the RecA-RecA interface found in RecA filaments. This would allow RecBCD to nucleate the formation of a RecA filament by depositing RecA protomers on the growing ssDNA loop following Chi recognition [1,81]. However, a recent electron microscopy reconstruction study has shown that the RecB nuclease domain binds to intact RecA filaments, which implies that the interaction between the two proteins cannot be made entirely with the RecA-RecA polymerisation interface [82]. Further work will be required to understand the details of RecA loading in RecBCD-type helicase–nucleases, which may have implications for our understanding of related mechanisms in human cells associated with the development of cancer [81]. 3. Mechanism of AddAB-type helicase–nucleases Just as had been the case in E. coli, a powerful ATP-dependent exonuclease activity was easily detectable in B. subtilis cells [83,84],

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and much of the early genetic and biochemical characterisation of AddAB-type complexes was performed on B. subtilis AddAB and L. lactis RexAB (reviewed in Refs. [14,85]). Importantly, in addition to establishing that AddAB and RecBCD are involved in similar cellular processes ([1,14,85] and Section 1.3) this work also showed that the purified enzymes display similar, but not identical, biochemical properties in vitro [20]. 3.1. Single motor–dual nuclease mechanism The B. subtilis AddAB complex exists as a 270 kDa heterodimer in free solution [86], and analytical ultracentrifugation and functional assays indicate that the enzyme also functions as a heterodimer of AddA and AddB (our unpublished data). The AddA subunit resembles RecB, in that it contains an N-terminal SF1 helicase domain and a C-terminal RecB-family nuclease domain (Fig. 4). The polarity of the helicase domain has never been determined experimentally but the primary structure is consistent with a 3 > 5 (SF1A) directionality [57]. Interestingly, the AddB subunit has weak homology to a SF1 helicase domain in the N-terminus [1,35]. Indeed, in many cases [35], this domain of AddB includes an intact WalkerA motif, which is equivalent to helicase motif I and is generally involved in ATP binding and hydrolysis. The AddB C-terminus encodes another RecB-family nuclease domain, which is homologous to those found in both AddA and RecB, but which (in many cases [35,86]) also contains four conserved cysteine residues that co-ordinate an iron–sulphur (Fe–S) cluster (Fig. 4; Section 3.3). Site-directed mutagenesis experiments have established that the AddA helicase is essential for helicase–nuclease activity, whereas interfering with the Walker-A motif in AddB seems to have little or no effect on the ability of the enzyme to unwind and degrade DNA ([87–89] and our unpublished data). Note that this contrasts with the situation for RecBCD: in that case either of the two motors can be inactivated without eliminating processive helicase activity [59,60,77]. The nuclease domains of AddA and AddB are both active [38,89–91], and they are each exclusively responsible for cleaving one of the nascent single-strands of DNA. AddA cleaves the 3 > 5 strand whereas AddB cuts the 5 > 3 strand [89]. Before Chi recognition, cleavage of the two single-strands is symmetric [20] suggesting that the two nuclease active sites operate at similar rates. The recognition of Chi results in cessation of cleavage on the 3 > 5 strand and, accordingly, it was found that the AddA nuclease domain was essential for the production of Chi-specific DNA fragments by AddAB and for recombination hotspot activity in vivo [89,90]. Taken together, these data suggest that, in distinct contrast to RecBCD, the AddAB-type helicase–nuclease uses a “single motor–dual nuclease” strategy for processive DNA end resection (Fig. 6). 3.2. Models for the interaction of AddAB with Chi sequences Recognition of Chi by AddAB elicits a similar response to that of RecBCD, in that cleavage of the 3 > 5 strand is attenuated to produce a recombinogenic 3 -ssDNA tail. It was shown that B. subtilis AddAB forms a very stable complex with its cognate Chi sequence [92], such that the enzyme can effectively be foot-printed at Chi following DNA processing [89]. This observation is consistent with the “Chi-scanning” model developed for RecBCD (Section 2.2). Indeed, the control of DNA cleavage by Chi may occur via a similar mechanism to that proposed for RecBCD, because the N-terminal domain of AddB appears to resemble an inactivated helicase domain as does RecC ([1,35,65]; Fig. 4). Moreover, the presence of an intact Walker-A motif in this region of many AddB proteins might imply a possible role for ATP binding in the stabilisation of the AddAB-Chi complex (Fig. 6), although this idea remains to be rigorously tested. The purpose of such a tightly-bound Chi complex is not understood

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3.3. An essential iron–sulphur cluster in B. subtilis AddAB

Fig. 6. Model for DNA end processing by an AddAB-type helicase–nuclease. (A) Proposed domain organisation of an AddAB-type helicase–nuclease. Before Chi recognition unwinding is driven by a single motor (red arrow) on the 3 > 5 strand. Each nascent single-strand encounters a separate nuclease domain (dark and light blue circles). (B) The observations that AddAB forms a stable complex with Chi and that AddB likely contains an inactivated helicase domain (Fig. 4), support a model in which the binding of Chi to the N-terminal domain of AddB (striped red arrow) prevents further hydrolysis of the 3 > 5 strand beyond Chi. As for RecBCD, a growing loop of ssDNA would form on the 3 > 5 strand after Chi recognition. Domain shapes and colours are as described in Figs 4 and 5.

at this time, but one might speculate that it promotes the loading of RecA protein onto the Chi-terminated single-stranded DNA overhang. RecA loading has never been directly demonstrated for an AddAB-type complex, although recent genetic complementation experiments with H. pylori AddAB and RecA are consistent with a RecA loading mechanism in that system [38]. In any case, the exceptional stability of the AddAB-Chi interaction may prove to be of practical use in future attempts to crystallise a complex between a helicase–nuclease and a recombination hotspot. Although no crystal structure is currently available for any AddAB-type complex, it is possible to make some informed predictions as to their general architecture and reaction mechanism (Fig. 6). The AddA helicase and nuclease domains presumably lie on the path of the 3 > 5 strand, in the same manner as the equivalent domains in RecB. If AddAB uses the same mechanism as RecBCD for Chi recognition then the helicase-like region of AddB must sit in between the helicase motor and the nuclease domain on the 3 > 5 strand. The active AddB nuclease domain must be located on the path of the 5 > 3 strand, perhaps in the equivalent position to the inactivated nuclease-like domain of RecC [89]. This organisation would allow AddAB to catalyse the same reaction as RecBCD using a different complement of catalytic domains, while retaining a similar overall architecture for the two classes of helicase–nuclease (Figs. 5 and 6). For the RecBCD enzymes, the physiological relevance of the cuts that are made in the 5 > 3 strand (and those made upstream of the Chi sequence on the 3 > 5 strand) has been the subject of some debate, and some models for recombination invoke a single “nick-at-Chi” to initiate strand exchange (for further details, discussions and contrasting opinions see Refs. [1,93,94]). However, in AddAB-type complexes, the presence of a dedicated nuclease domain for cleavage of the 5 > 3 strand strongly suggests that cuts are indeed made on the 5 > 3 strand in vivo, at least in organisms that employ an AddAB-type complex for DSB repair. Indeed, both nuclease domains of AddAB were shown to contribute to DNA damage tolerance and infectivity in H. pylori [38].

A further distinction between AddAB and RecBCD was uncovered with the unexpected finding that many AddAB-type complexes contain an Fe–S cluster [86]. Using electron paramagnetic resonance spectroscopy, it was shown that B. subtilis AddAB contains a cubane 3Fe–4S or 4Fe–4S cluster. Four cysteine residues were shown to be important for its co-ordination. These form an unusual arrangement of ligands flanking both sides of the nuclease domain (Fig. 4). Because these ligands were all required for the localised structural integrity of the AddB nuclease, this region of AddB was named an “iron-staple” nuclease domain. Loss of the cluster completely eliminates the ability of AddAB to bind to duplex DNA ends and DNA end binding has a strong stabilising effect on the Fe–S cluster, suggesting that the cluster plays either a direct or indirect role in binding to DSBs. The XPD-like helicases involved in nucleotide excision repair also contain an Fe–S cluster [95], which may function as a “pin” [96]: a common structural element in helicases which assists in the separation of duplex DNA at the ss–dsDNA junction [55]. It is conceivable that the cluster in AddAB fulfils an equivalent role because complexes with mutations in the Fe–S ligands retain the ability to translocate on ssDNA [86]. Despite a recent rush of publications, Fe–S clusters remain relatively rare in DNA binding proteins and their possible roles are not well-characterised [97]. Notably, Fe–S clusters were also discovered in certain families of DNA glycosylases that include EndoIII and MutY [98,99]. Because those clusters are redox active under physiological conditions when the enzymes are bound to DNA, it was proposed that DNA-mediated charge transfer may play a role in the detection of DNA damage (discussed in Refs. [100,101]). However, in AddAB, the Fe–S cluster is common but by no means ubiquitous [35] and is clearly not required in the functionally analogous RecBCD-type complexes. In our opinion, this observation argues against any essential function of AddAB involving electron transfer. Interestingly, some bacterial species that apparently encode iron-free AddAB complexes are also able to tolerate exceptionally low intracellular iron concentrations [102,103]. This may reflect an evolutionary adaption to avoid the damaging effects of reactive oxygen species that are produced by the Fenton reaction for which iron is a catalyst [104,105]. Sinha et al. recently characterised AdnAB: a DNA end resection enzyme from Mycobacterium smegmatis which, interestingly, also possesses a RecBCD complex [106]. AdnAB displays a similar overall primary structure to AddAB, containing one conventional RecBfamily nuclease domain and one “iron-staple” nuclease domain. However, it was argued that AdnAB was distinct from both RecBCDand AddAB-type helicase–nucleases in that it apparently displays a dual motor–dual nuclease organisation [106,107]. However, the DNA motor activity of AdnA (equivalent to AddB) was inferred solely on the basis of partially conserved helicase motifs, and this putative activity was not demonstrated experimentally. Given the likelihood that both RecC and AddB employ helicase-like domains in a non-standard capacity as a sequence recognition element, it remains possible that the AdnA “helicase” plays the same role in AdnAB. In this regard, it will be important to establish if the AdnA protein is in fact a bona fide DNA motor and also whether AdnAB recognises and responds to a Chi sequence. 4. Comparison between bacterial helicase–nuclease mechanisms Helicase–nucleases provide fascinating model systems for understanding how the DNA helicase motor can be integrated with other protein machinery. Built of simple components, but more than the sum of their parts, their design illustrates principles of modularity in the origin of complex DNA processing reactions. Furthermore, they are unique amongst DNA helicases in being con-

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trolled upon encounter with their specific Chi sequences in cis. The recognition of Chi also illustrates how nature has modified the helicase domain (a single-stranded DNA motor) and redeployed it in a new role as a scanner for specific DNA sequences, highlighting the remarkable functional plasticity of “helicase” domains [55,108]. A comparison of the two classes of helicase–nuclease reveals several common themes and important differences. Fundamentally, AddAB and RecBCD catalyse the same reaction and largely perform the same role in cells, as demonstrated by their ability to substitute for each other in vivo. Both are highly processive helicases and voracious nucleases that are controlled by encounter with specific (but different) single-stranded DNA sequences. Both enzyme classes are constructed from a combination of catalytically active and inactive helicase and nuclease domains, but different mechanistic strategies are used to achieve the same goal. Exactly what costs and benefits are associated with each design is still unclear. Presumably the bipolar motor organisation of RecBCD is energetically more costly than the simpler single motor arrangement in AddAB. However, this might increase the rate, processivity and force generation of the translocating enzyme [77] or allow the complex to bypass DNA damage in both strands of the duplex [59]. Many other questions remain unanswered, including why many AddAB-type enzymes contain an apparently intact Walker-A motif and/or an Fe–S cluster. Furthermore, relatively little is known about how DNA end resection is co-ordinated with upstream (replication fork collapse or DSB formation) and downstream (strand exchange) steps in the repair pathway. Although AddAB and RecBCD can both handle some variation in the structure of the DNA end, substantial ssDNA overhangs and hairpins eliminate their ability to initiate DNA unwinding [16,20,109]. The precise structures of DSBs formed in vivo under physiologically relevant conditions are not well understood [7] but it seems likely that the AddAB and RecBCD enzymes may be required to function on a wider variety of broken DNA substrates than their in vitro properties would suggest. Consequently, other DNA end processing and tethering factors that play early roles in DSB repair may be important in pre-processing and organising DSBs containing overhangs, secondary structures, hairpins or cross-linked proteins that are not amenable to binding by AddAB/RecBCD [110,111]. Similar factors might also help to explain how DSBs are channelled into the appropriate HR or NHEJ repair pathway, or how DSB repair is co-ordinated with DNA dynamics and cell division [112]. 5. Emerging parallels between bacterial and eukaryotic DNA end resection? In eukaryotes, the repair of DSBs by HR is also initiated by their conversion into 3 -ssDNA tails that are substrates for Rad51mediated strand exchange [113–115]. However, until very recently, our understanding of the protein factors responsible for DNA end resection had been very poorly defined. This situation was turned on its head by an abundance of recent work that has now identified multiple helicase and nuclease activities involved in the resection of DNA ends (for reviews see Refs. [107,116–118]). The MRX/MRN complex had long been implicated in the initiation of recombination, but its biochemical properties were not commensurate with it being responsible for extensive DNA resection with the appropriate nuclease polarity [119,120]. It now appears that MRX/MRN, in conjunction with the Sae2/CtIP nuclease, may be involved in the initial degradation and spatial organisation of DNA ends, whereas extensive resection is promoted by Sgs1/BLM (a SF2A helicase of the RecQ-family), Exo1 (a 5 > 3 dsDNA exonuclease) and Dna2 (an N-terminal RecB-family nuclease fused to a SF1B helicase domain) [121–129]. Although speculative at this stage, it looks increasingly as if the eukaryotic DNA end resection machinery may at least partially resemble the prokaryotic systems. The MRX/MRN complex

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is structurally related to the prokaryotic SbcCD complex and RecN protein (both prokaryotic SMC-family proteins) which might also organise and prepare DSBs for extensive resection by other proteins (Fig. 1). Sgs1/BLM and Exo1 may be functionally analogous to prokaryotic RecQ (a SF2A helicase) and RecJ (a 5 > 3 ssDNA and dsDNA exonuclease), proteins which are known to provide back-up for the processing of DSBs by AddAB/RecBCD (Fig. 1, [4,110]). This idea is supported by in vitro reconstitution of the initial steps of DSB repair using bacterial and eukaryotic proteins [129,130]. Finally, although there are no structural homologues of AddAB/RecBCD and no evidence for a Chi-like recombination hotspot in eukaryotic cells, parallels had previously been drawn between the general domain architecture of the RecBCD helicase–nuclease and the Dna2 helicase–nuclease [131]. In the light of new data implicating Dna2 in DNA end resection, we were interested to discover that the N-terminal nuclease domain of Dna2 contains exactly the same cysteine motifs as those present in AddAB, strongly suggesting that Dna2 also contains a rare iron-staple nuclease domain [86]. Unfortunately, there is relatively little published data on the biochemical properties of Dna2 protein, which may well reflect the presence of an unstable Fe–S cluster in the purified protein. In all kingdoms of life, exactly how all of the available helicase and nuclease activities are orchestrated to process DSBs in vivo remains to be seen. It seems likely that the complexity of the protein machinery might reflect the need to channel a variety of different DNA end structures, or DNA–protein cross-links, into a choice of different repair pathways. This will be an exciting area for future research using a combination of genetic and biochemical methods. Conflict of interest The authors have no conflicts of interest relating to the publication of this article. Acknowledgments We are grateful to Dr Emma Gwynn for her comments on the manuscript, and to Drs Steve Kowalczykowski and Dale Wigley for many stimulating discussions regarding the mechanisms of bacterial helicase–nuclease complexes. Work in the author’s laboratory is supported by the Royal Society, the Biotechnology and Biological Sciences Research Council, and the European Research Council. References [1] M.S. Dillingham, S.C. Kowalczykowski, RecBCD enzyme and the repair of double-stranded DNA breaks, Microbiol. Mol. Biol. Rev. 72 (2008) 642–671, Table of Contents. [2] A. Kuzminov, Collapse and repair of replication forks in Escherichia coli, Mol. Microbiol. 16 (1995) 373–384. [3] J. Atkinson, P. McGlynn, Replication fork reversal and the maintenance of genome stability, Nucleic Acids Res. 37 (2009) 3475–3492. [4] M. Spies, S.C. Kowalczykowski, Homologous Recombination By The RecBCD and RecF pathways, in: The Bacterial Chromosome, ASM press, Washington, D.C., 2005, pp. 389–403. [5] S.C. Kowalczykowski, Initiation of genetic recombination and recombinationdependent replication, Trends Biochem. Sci. 25 (2000) 156–165. [6] B. Michel, H. Boubakri, Z. Baharoglu, M. LeMasson, R. Lestini, Recombination proteins and rescue of arrested replication forks, DNA Repair (Amst) 6 (2007) 967–980. [7] M.M. Cox, Recombinational DNA repair of damaged replication forks in Escherichia coli: questions, Annu. Rev. Genet. 35 (2001) 53–82. [8] S.C. Kowalczykowski, D.A. Dixon, A.K. Eggleston, S.D. Lauder, W.M. Rehrauer, Biochemistry of homologous recombination in Escherichia coli, Microbiol. Rev. 58 (1994) 401–465. [9] H. Sanchez, B. Carrasco, S. Ayora, J.C. Alonso, Homologous Recombination in Low dC + dG Gram-Positive Bacteria, in: A. Aguilera, R. Rothstein (Eds.), Molecular Genetics of Recombination, Springer, Berlin/Heidelberg, 2007, pp. 27–52. [10] R. Bowater, A.J. Doherty, Making ends meet: repairing breaks in bacterial DNA by non-homologous end joining, PLoS Genet. 2 (2006) e8.

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