Structural and functional analyses of an archaeal XPF/Rad1/Mus81 nuclease: asymmetric DNA binding and cleavage mechanisms

XPF/Rad1/Mus81/Hef proteins recognize and cleave branched DNA structures. XPF and Rad1 proteins cleave the 5' side of nucleotide excision repair bubble, while Mus81 and Hef cleave similar sites of the nicked Holliday junction, fork, or flap structure. These proteins all function as dimers and consist of catalytic and helix-hairpin-helix DNA binding (HhH) domains. We have determined the crystal structure of the HhH domain of Pyrococcus furiosus Hef nuclease (HefHhH), which revealed the distinct mode of protein dimerization. Our structural and biochemical analyses also showed that each of the catalytic and HhH domains binds to distinct regions within the fork-structured DNA: each HhH domain from two separate subunits asymmetrically binds to the arm region, while the catalytic domain binds near the junction center. Upon binding to DNA, Hef nuclease disrupts base pairs near the cleavage site. It is most likely that this bipartite binding mode is conserved in the XPF/Rad1/Mus81 nuclease family.     

Cooperation of the N-terminal Helicase and C-terminal endonuclease activities of Archaeal Hef protein in processing stalled replication forks

Blockage of replication fork progression often occurs during DNA replication, and repairing and restarting stalled replication forks are essential events in all organisms for the maintenance of genome integrity. The repair system employs processing enzymes to restore the stalled fork. In Archaea Hef is a well conserved protein that specifically cleaves nicked, flapped, and fork-structured DNAs. This enzyme contains two distinct domains that are similar to the DEAH helicase family and XPF nuclease superfamily proteins. Analyses of truncated mutant proteins consisting of each domain revealed that the C-terminal nuclease domain independently recognized and incised fork-structured DNA. The N-terminal helicase domain also specifically unwound fork-structured DNA and Holliday junction DNA in the presence of ATP. Moreover, the endonuclease activity of the whole Hef protein was clearly stimulated by ATP hydrolysis catalyzed by the N-terminal domain. These enzymatic properties suggest that Hef efficiently resolves stalled replication forks by two steps, which are branch point transfer to the 5'-end of the nascent lagging strand by the N-terminal helicase followed by template strand incision for leading strand synthesis by the C-terminal endonuclease.     

X-ray and biochemical anatomy of an archaeal XPF/Rad1/Mus81 family nuclease: similarity between its endonuclease domain and restriction enzymes

The XPF/Rad1/Mus81-dependent nuclease family specifically cleaves branched structures generated during DNA repair, replication, and recombination, and is essential for maintaining genome stability. Here, we report the domain organization of an archaeal homolog (Hef) of this family and the X-ray crystal structure of the middle domain, with the nuclease activity. The nuclease domain architecture exhibits remarkable similarity to those of restriction endonucleases, including the correspondence of the GDX(n)ERKX(3)D signature motif in Hef to the PDX(n)(E/D)XK motif in restriction enzymes. This structural study also suggests that the XPF/Rad1/Mus81/ERCC1 proteins form a dimer through each interface of the nuclease domain and the helix-hairpin-helix domain. Simultaneous disruptions of both interfaces result in their dissociation into separate monomers, with strikingly reduced endonuclease activities.     

The archaeal Xpf/Mus81/FANCM homolog Hef and the Holliday junction resolvase Hjc define alternative pathways that are essential for cell viability in Haloferax volcanii

The XPF/MUS81 family of endonucleases is found in eukaryotes and archaea, in the former they play a critical role in DNA repair and replication fork restart. Hef is a XPF/MUS81 family member found in Euryarchaea and is related to the Fanconi anemia protein FANCM. We have studied the role of Hef in the euryarchaeon Haloferax volcanii. Unlike Xpf in eukaryotes, Hef is not involved in nucleotide excision repair; instead, this function is encoded by the uvrABC genes. Similarly, deletion of hef confers only moderate sensitivity to DNA crosslinking agents, whereas mutation of FANCM in leads to hypersensitivity in eukaryotes. However, Hef is essential for cell viability when the Holliday junction resolvase Hjc is absent, and both the helicase and nuclease activities of Hef are indispensable. By contrast, single mutants of hjc and hef display no significant defects in growth or homologous recombination. This suggests that Hef and Hjc are redundant for the resolution of recombination intermediates, and that Hef is the functional homolog of eukaryotic Mus81. Furthermore, deletion of hef in a recombination-deficient ΔradA background is highly deleterious but deletion of hjc has no effect. Therefore, Hjc acts exclusively in homologous recombination whereas Hef, in addition to its role in resolving recombination intermediates, can act in a pathway that avoids the use of homologous recombination. We propose that Hef and Hjc provide alternative means to restart stalled DNA replication forks.     

Novel endonuclease in Archaea cleaving DNA with various branched structure

We identified a novel structure-specific endonuclease in Pyrococcus furiosus. This nuclease contains two distinct domains, which are similar to the DEAH helicase family at the N-terminal two-third and the XPF endonuclease superfamily at the C-terminal one-third of the protein, respectively. The C-terminal domain has an endonuclease activity cleaving the DNA strand at the 5'-side of nicked or flapped positions in the duplex DNA. The nuclease also incises in the proximity of the 5'-side of a branch point in the template strand for leading synthesis in the fork-structured DNA. The N-terminal helicase may work cooperatively to change the fork structure suitable for cleavage by the C-terminal endonuclease. This protein, designated as Hef (helicase-associated endonuclease for fork-structured DNA), may be a prototypical enzyme for resolving stalled forks during DNA replication, as well as working at nucleotide excision repair.     

Intracellular dynamics of archaeal FANCM homologue Hef in response to halted DNA replication

Hef is an archaeal member of the DNA repair endonuclease XPF (XPF)/Crossover junction endonuclease MUS81 (MUS81)/Fanconi anemia, complementation group M (FANCM) protein family that in eukaryotes participates in the restart of stalled DNA replication forks. To investigate the physiological roles of Hef in maintaining genome stability in living archaeal cells, we studied the localization of Hef–green fluorescent protein fusions by fluorescence microscopy. Our studies revealed that Haloferax volcanii Hef proteins formed specific localization foci under regular growth conditions, the number of which specifically increased in response to replication arrest. Purification of the full-length Hef protein from its native host revealed that it forms a stable homodimer in solution, with a peculiar elongated configuration. Altogether our data indicate that the shape of Hef, significant physicochemical constraints and/or interactions with DNA limit the apparent cytosolic diffusion of halophilic DNA replication/repair complexes, and demonstrate that Hef proteins are dynamically recruited to archaeal eukaryotic-like chromatin to counteract DNA replication stress. We suggest that the evolutionary conserved function of Hef/FANCM proteins is to enhance replication fork stability by directly interacting with collapsed replication forks.     

The active site of the DNA repair endonuclease XPF-ERCC1 forms a highly conserved nuclease motif

XPF-ERCC1 is a structure-specific endonuclease involved in nucleotide excision repair, interstrand crosslink repair and homologous recombination. So far, it has not been shown experimentally which subunit of the heterodimer harbors the nuclease activity and which amino acids contribute to catalysis. We used an affinity cleavage assay and located the active site to amino acids 670-740 of XPF. Point mutations generated in this region were analyzed for their role in nuclease activity, metal coordination and DNA binding. Several acidic and basic residues turned out to be required for nuclease activity, but not DNA binding. The separation of substrate binding and catalysis by XPF-ERCC1 will be invaluable in studying the role of this protein in various DNA repair processes. Alignment of the active site region of XPF with proteins belonging to the Mus81 family and a putative archaeal RNA helicase family reveals that seven of the residues of XPF involved in nuclease activity are absolutely conserved in the three protein families, indicating that they share a common nuclease motif.     

Analysis of the XPA and ssDNA-binding surfaces on the central domain of human ERCC1 reveals evidence for subfunctionalization

Human ERCC1/XPF is a structure-specific endonuclease involved in multiple DNA repair pathways. We present the solution structure of the non-catalytic ERCC1 central domain. Although this domain shows structural homology with the catalytically active XPF nuclease domain, functional investigation reveals a completely distinct function for the ERCC1 central domain by performing interactions with both XPA and single-stranded DNA. These interactions are non-competitive and can occur simultaneously through distinct interaction surfaces. Interestingly, the XPA binding by ERCC1 and the catalytic function of XPF are dependent on a structurally homologous region of the two proteins. Although these regions are strictly conserved in each protein family, amino acid composition and surface characteristics are distinct. We discuss the possibility that after XPF gene duplication, the redundant ERCC1 central domain acquired novel functions, thereby increasing the fidelity of eukaryotic DNA repair.     

Identification of FAAP24, a Fanconi anemia core complex protein that interacts with FANCM

The Fanconi anemia (FA) core complex plays a crucial role in a DNA damage response network with BRCA1 and BRCA2. How this complex interacts with damaged DNA is unknown, as only the FA core protein FANCM (the homolog of an archaeal helicase/nuclease known as HEF) exhibits DNA binding activity. Here, we describe the identification of FAAP24, a protein that targets FANCM to structures that mimic intermediates formed during the replication/repair of damaged DNA. FAAP24 shares homology with the XPF family of flap/fork endonucleases, associates with the C-terminal region of FANCM, and is a component of the FA core complex. FAAP24 is required for normal levels of FANCD2 monoubiquitylation following DNA damage. Depletion of FAAP24 by siRNA results in cellular hypersensitivity to DNA crosslinking agents and chromosomal instability. Our data indicate that the FANCM/FAAP24 complex may play a key role in recruitment of the FA core complex to damaged DNA.     

The structure of the human ERCC1/XPF interaction domains reveals a complementary role for the two proteins in nucleotide excision repair

The human ERCC1/XPF complex is a structure-specific endonuclease with defined polarity that participates in multiple DNA repair pathways. We report the heterodimeric structure of the C-terminal domains of both proteins responsible for ERCC1/XPF complex formation. Both domains exhibit the double helix-hairpin-helix motif (HhH)2, and they are related by a pseudo-2-fold symmetry axis. In the XPF domain, the hairpin of the second motif is replaced by a short turn. The ERCC1 domain folds properly only in the presence of the XPF domain, which implies a role for XPF as a scaffold for the folding of ERCC1. The intersubunit interactions are largely hydrophobic in nature. NMR titration data show that only the ERCC1 domain of the ERCC1/XPF complex is involved in DNA binding. On the basis of these findings, we propose a model for the targeting of XPF nuclease via ERCC1-mediated interactions in the context of nucleotide excision repair.     

Structure of an XPF endonuclease with and without DNA suggests a model for substrate recognition

The XPF/Mus81 structure-specific endonucleases cleave double-stranded DNA (dsDNA) within asymmetric branched DNA substrates and play an essential role in nucleotide excision repair, recombination and genome integrity. We report the structure of an archaeal XPF homodimer alone and bound to dsDNA. Superposition of these structures reveals a large domain movement upon binding DNA, indicating how the (HhH)(2) domain and the nuclease domain are coupled to allow the recognition of double-stranded/single-stranded DNA junctions. We identify two nonequivalent DNA-binding sites and propose a model in which XPF distorts the 3' flap substrate in order to engage both binding sites and promote strand cleavage. The model rationalises published biochemical data and implies a novel role for the ERCC1 subunit of eukaryotic XPF complexes.     

DNA end-directed and processive nuclease activities of the archaeal XPF enzyme

The XPF/Mus81 family of structure-specific nucleases cleaves branched or nicked DNA substrates and are implicated in a wide range of DNA repair and recombination processes. The structure of the crenarchaeal XPF bound to a DNA duplex has revealed a plausible mechanism for DNA binding, involving DNA distortion into upstream and downstream duplexes engaged by the two helix–hairpin–helix domains that form a dimeric structure at the C-terminus of the enzyme. A flexible linker joins these to the dimeric nuclease domain, and a C-terminal motif interacts with the sliding clamp, which is essential for the activity of the enzyme. Here, we demonstrate the importance of the downstream duplex in directing the endonuclease activity of crenarchaeal XPF, which is similar to that of Mus81-Eme1, and suggest a mechanistic basis for this control. Furthermore, our data reveal that the enzyme can digest a nicked DNA strand processively over at least 60 nt in a 3′–5′ direction and can remove varied types of DNA lesions and blocked DNA termini. This in vitro activity suggests a potential role for crenarchaeal XPF in a variety of repair processes for which there are no clear pathways in archaea.     

The Walker B motif in avian FANCM is required to limit sister chromatid exchanges but is dispensable for DNA crosslink repair

FANCM, the most highly conserved component of the Fanconi Anaemia (FA) pathway can resolve recombination intermediates and remodel synthetic replication forks. However, it is not known if these activities are relevant to how this conserved protein activates the FA pathway and promotes DNA crosslink repair. Here we use chicken DT40 cells to systematically dissect the function of the helicase and nuclease domains of FANCM. Our studies reveal that these domains contribute distinct roles in the tolerance of crosslinker, UV light and camptothecin-induced DNA damage. Although the complete helicase domain is critical for crosslink repair, a predicted inactivating mutation of the Walker B box domain has no impact on FA pathway associated functions. However, this mutation does result in elevated sister chromatid exchanges (SCE). Furthermore, our genetic dissection indicates that FANCM functions with the Blm helicase to suppress spontaneous SCE events. Overall our results lead us to reappraise the role of helicase domain associated activities of FANCM with respect to the activation of the FA pathway, crosslink repair and in the resolution of recombination intermediates.     

Crystal structure of the hexameric replicative helicase RepA of plasmid RSF1010

Unwinding of double-stranded DNA into single-stranded intermediates required for various fundamental life processes is catalyzed by helicases, a family of mono-, di- or hexameric motor proteins fueled by nucleoside triphosphate hydrolysis. The three-dimensional crystal structure of the hexameric helicase RepA encoded by plasmid RSF1010 has been determined by X-ray diffraction at 2.4 A resolution. The hexamer shows an annular structure with 6-fold rotational symmetry and a approximately 17 A wide central hole, suggesting that single-stranded DNA may be threaded during unwinding. Homologs of all five conserved sequence motifs of the DnaB-like helicase family are found in RepA, and the topography of the monomer resembles RecA and the helicase domain of the bacteriophage T7 gp4 protein. In a modeled complex, ATP molecules are located at the subunit interfaces and clearly define adenine-binding and ATPase catalytic sites formed by amino acid residues located on adjacent monomers; most remarkable is the "arginine finger" Arg207 contributing to the active site in the adjacent monomer. This arrangement of active-site residues suggests cooperativity between monomers in ATP hydrolysis and helicase activity of RepA. The mechanism of DNA unwinding remains elusive, as RepA is 6-fold symmetric, contrasting the recently published asymmetric structure of the bacteriophage T7 gp4 helicase domain.     

Staphylococcus aureus DinG, a helicase that has evolved into a nuclease

DinG (damage inducible gene G) is a bacterial superfamily 2 helicase with 5′→3′ polarity. DinG is related to the XPD (xeroderma pigmentosum complementation group D) helicase family, and they have in common an FeS (iron–sulfur)-binding domain that is essential for the helicase activity. In the bacilli and clostridia, the DinG helicase has become fused with an N-terminal domain that is predicted to be an exonuclease. In the present paper we show that the DinG protein from Staphylococcus aureus lacks an FeS domain and is not a DNA helicase, although it retains DNA-dependent ATP hydrolysis activity. Instead, the enzyme is an active 3′→5′ exonuclease acting on single-stranded DNA and RNA substrates. The nuclease activity can be modulated by mutation of the ATP-binding cleft of the helicase domain, and is inhibited by ATP or ADP, suggesting a modified role for the inactive helicase domain in the control of the nuclease activity. By degrading rather than displacing RNA or DNA strands, the S. aureus DinG nuclease may accomplish the same function as the canonical DinG helicase.     

Physical and functional interaction between the XPF/ERCC1 endonuclease and hRad52

The XPF/ERCC1 heterodimer is a DNA structure-specific endonuclease that participates in nucleotide excision repair and homology-dependent recombination reactions, including DNA single strand annealing and gene targeting. Here we show that XPF/ERCC1 is stably associated with hRad52, a recombinational repair protein, in human cell-free extracts and that these factors interact directly via the N-terminal domain of hRad52 and the XPF protein. Complex formation between hRad52 and XPF/ERCC1 concomitantly stimulates the DNA structure-specific endonuclease activity of XPF/ERCC1 and attenuates the DNA strand annealing activity of hRad52. Our results reveal a novel role for hRad52 as a subunit of a DNA structure-specific endonuclease and are congruent with evidence implicating both hRad52 and XPF/ERCC1 in a number of homologous recombination reactions. We propose that the ternary complex of hRad52 and XPF/ERCC1 is the active species that processes recombination intermediates generated during the repair of DNA double strand breaks and in homology-dependent gene targeting events.     

Recruitment and positioning determine the specific role of the XPF‐ERCC1 endonuclease in interstrand crosslink repair

XPF‐ERCC1 is a structure‐specific endonuclease pivotal for several DNA repair pathways and, when mutated, can cause multiple diseases. Although the disease‐specific mutations are thought to affect different DNA repair pathways, the molecular basis for this is unknown. Here we examine the function of XPF‐ERCC1 in DNA interstrand crosslink (ICL) repair. We used Xenopus egg extracts to measure both ICL and nucleotide excision repair, and we identified mutations that are specifically defective in ICL repair. One of these separation‐of‐function mutations resides in the helicase‐like domain of XPF and disrupts binding to SLX4 and recruitment to the ICL. A small deletion in the same domain supports recruitment of XPF to the ICL, but inhibited the unhooking incisions most likely by disrupting a second, transient interaction with SLX4. Finally, mutation of residues in the nuclease domain did not affect localization of XPF‐ERCC1 to the ICL but did prevent incisions on the ICL substrate. Our data support a model in which the ICL repair‐specific function of XPF‐ERCC1 is dependent on recruitment, positioning and substrate recognition.     

Domain mapping of the DNA binding, endonuclease, and ERCC1 binding properties of the human DNA repair protein XPF.

During nucleotide excision repair, one of the two incisions necessary for removal of a broad spectrum of DNA adducts is made by the human XPF/ERCC1 protein complex. To characterize the biochemical function of XPF, we have expressed and purified the independent 104 kDa recombinant XPF protein from E. coli and determined that it is an endonuclease and can bind DNA in the absence of the ERCC1 subunit. Endonuclease activity was also identified in a stable 70 kDa proteolysis fragment of XPF obtained during protein expression, indicating an N-terminal catalytic domain. Sequence homology and secondary structure predictions indicated a second functional domain at the C-terminus of XPF. To investigate the significance of the two predicted domains, a series of XPF deletion fragments spanning the entire protein were designed and examined for DNA binding, endonuclease activity, and ERCC1 subunit binding. Our results indicate that the N-terminal 378 amino acids of XPF are capable of binding and hydrolyzing DNA, while the C-terminal 214 residues are capable of binding specifically to ERCC1. We propose that the N-terminal domain of XPF contributes to the junction-specific endonuclease activity observed during DNA repair and recombination events. In addition, evidence presented here suggests that the C-terminal domain of XPF is responsible for XPF/ERCC1 complex formation. A working model for the XPF protein is presented illustrating the function of XPF in the nucleotide excision pathway and depicting the two functional domains interacting with DNA and ERCC1.     

Activity of individual ERCC1 and XPF subunits in DNA nucleotide excision repair

ERCC1–XPF is a structure-specific nuclease with two subunits, ERCC1 and XPF. The enzyme cuts DNA at junctions where a single strand moves 5′ to 3′ away from a branch point with duplex DNA. This activity has a central role in nucleotide excision repair (NER), DNA cross-link repair and recombination. To dissect the activities of the nuclease it is necessary to investigate the subunits individually, as studies of the enzyme so far have only used the heterodimeric complex. We produced recombinant ERCC1 and XPF separately in Escherichia coli as soluble proteins. Activity was monitored by a sensitive dual incision assay for NER by complementation of cell extracts. XPF and ERCC1 are unstable in mammalian cells in the absence of their partners but we found, surprisingly, that ERCC1 alone could confer some repair to extracts from ERCC1-defective cells. A version of ERCC1 lacking the first 88 non-conserved amino acids was also functional. This indicated that a small amount of active XPF was present in ERCC1 extracts, and immunoassays showed this to be the case. Some repair in XPF-defective extracts could be achieved by adding ERCC1 and XPF proteins together, but not by adding only XPF. The results show for the first time that functional ERCC1–XPF can be formed from separately produced subunits. Protein sequence comparison revealed similarity between the ERCC1 family and the C-terminal region of the XPF family, including the regions of both proteins that are necessary for the ERCC1–XPF heterodimeric interaction. This suggests that the ERCC1 and XPF families are related via an ancient duplication.