Strikingly High Activity of 15-Lipoxygenase Towards Di-Polyunsaturated Arachidonoyl/Adrenoyl-Phosphatidylethanolamines Generates Peroxidation Signals of Ferroptotic Cell Death.

The vast majority of membrane phospholipids (PLs) include two asymmetrically positioned fatty acyls: oxidizable polyunsaturated fatty acids (PUFA) attached predominantly at the sn2 position, and non-oxidizable saturated/monounsaturated acids (SFA/MUFA) localized at the sn1 position. The peroxidation of PUFA-PLs, particularly sn2-arachidonoyl(AA)- and sn2-adrenoyl(AdA)-containing phosphatidylethanolamines (PE), has been associated with the execution of ferroptosis, a program of regulated cell death. There is a minor subpopulation (≈1-2 mol %) of doubly PUFA-acylated phospholipids (di-PUFA-PLs) whose role in ferroptosis remains enigmatic. Here we report that 15-lipoxygenase (15LOX) exhibits unexpectedly high pro-ferroptotic peroxidation activity towards di-PUFA-PEs. We revealed that peroxidation of several molecular species of di-PUFA-PEs occurred early in ferroptosis. Ferrostatin-1, a typical ferroptosis inhibitor, effectively prevented peroxidation of di-PUFA-PEs. Furthermore, co-incubation of cells with di-AA-PE and 15LOX produced PUFA-PE peroxidation and induced ferroptotic death. The decreased contents of di-PUFA-PEs in ACSL4 KO A375 cells was associated with lower levels of di-PUFA-PE peroxidation and enhanced resistance to ferroptosis. Thus, di-PUFA-PE species are newly identified phospholipid peroxidation substrates and regulators of ferroptosis, representing a promising therapeutic target for many diseases related to ferroptotic death.

Membrane regulation of 15LOX-1/PEBP1 complex prompts the generation of ferroptotic signals, oxygenated PEs.

Ferroptosis is a regulated form of cell death, the mechanism of which is still to be understood. 15-lipoxygenase (15LOX) complex with phosphatidylethanolamine (PE)-binding protein 1 (PEBP1) catalyzes the generation of pro-ferroptotic cell death signals, hydroperoxy-polyunsaturated PE. We focused on gaining new insights into the molecular basis of these pro-ferroptotic interactions using computational modeling and liquid chromatography-mass spectrometry experiments. Simulations of 15LOX-1/PEBP1 complex dynamics and interactions with lipids revealed that association with the membrane triggers a conformational change in the complex. This conformational change facilitates the access of stearoyl/arachidonoyl-PE (SAPE) substrates to the catalytic site. Furthermore, the binding of SAPE promotes tight interactions within the complex and induces further conformational changes that facilitate the oxidation reaction. The reaction yields two hydroperoxides as products, 15-HpETE-PE and 12-HpETE-PE, at a ratio of 5:1. A significant effect of PEBP1 is observed only on the predominant product. Moreover, combined experiments and simulations consistently demonstrate the significance of PEBP1 P112E mutation in generating ferroptotic cell death signals.

The Cytotoxic Mycobacteriophage Protein Phaedrus gp82 Interacts with and Modulates the Activity of the Host ATPase, MoxR.

Approximately 70% of bacteriophage-encoded proteins are of unknown function. Elucidating these protein functions represents opportunities to discover new phage-host interactions and mechanisms by which the phages modulate host activities. Here, we describe a pipeline for prioritizing phage-encoded proteins for structural analysis and characterize the gp82 protein encoded by mycobacteriophage Phaedrus. Structural and solution studies of gp82 show it is a trimeric protein containing two domains. Co-precipitation studies with the host Mycobacterium smegmatis identified the ATPase MoxR as an interacting partner protein. Phaedrus gp82-MoxR interaction requires the presence of a loop sequence within gp82 that is highly exposed and disordered in the crystallographic structure. We show that Phaedrus gp82 overexpression in M. smegmatis retards the growth of M. smegmatis on solid medium, resulting in a small colony phenotype. Overexpression of gp82 containing a mutant disordered loop or the overexpression of MoxR both rescue this phenotype. Lastly, we show that recombinant gp82 reduces levels of MoxR-mediated ATPase activity in vitro that is required for its chaperone function, and that the disordered loop plays an important role in this phenotype. We conclude that Phaedrus gp82 binds to and reduces mycobacterial MoxR activity, leading to reduced function of host proteins that require MoxR chaperone activity for their normal activity.

Discovering selective antiferroptotic inhibitors of the 15LOX/PEBP1 complex noninterfering with biosynthesis of lipid mediators.

Programmed ferroptotic death eliminates cells in all major organs and tissues with imbalanced redox metabolism due to overwhelming iron-catalyzed lipid peroxidation under insufficient control by thiols (Glutathione (GSH)). Ferroptosis has been associated with the pathogenesis of major chronic degenerative diseases and acute injuries of the brain, cardiovascular system, liver, kidneys, and other organs, and its manipulation offers a promising new strategy for anticancer therapy. This explains the high interest in designing new small-molecule-specific inhibitors against ferroptosis. Given the role of 15-lipoxygenase (15LOX) association with phosphatidylethanolamine (PE)-binding protein 1 (PEBP1) in initiating ferroptosis-specific peroxidation of polyunsaturated PE, we propose a strategy of discovering antiferroptotic agents as inhibitors of the 15LOX/PEBP1 catalytic complex rather than 15LOX alone. Here we designed, synthesized, and tested a customized library of 26 compounds using biochemical, molecular, and cell biology models along with redox lipidomic and computational analyses. We selected two lead compounds, FerroLOXIN-1 and 2, which effectively suppressed ferroptosis in vitro and in vivo without affecting the biosynthesis of pro-/anti-inflammatory lipid mediators in vivo. The effectiveness of these lead compounds is not due to radical scavenging or iron-chelation but results from their specific mechanisms of interaction with the 15LOX-2/PEBP1 complex, which either alters the binding pose of the substrate [eicosatetraenoyl-PE (ETE-PE)] in a nonproductive way or blocks the predominant oxygen channel thus preventing the catalysis of ETE-PE peroxidation. Our successful strategy may be adapted to the design of additional chemical libraries to reveal new ferroptosis-targeting therapeutic modalities.

Protein-metabolite interactomics of carbohydrate metabolism reveal regulation of lactate dehydrogenase.

Metabolic networks are interconnected and influence diverse cellular processes. The protein-metabolite interactions that mediate these networks are frequently low affinity and challenging to systematically discover. We developed mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) to identify such interactions. Analysis of 33 enzymes from human carbohydrate metabolism identified 830 protein-metabolite interactions, including known regulators, substrates, and products as well as previously unreported interactions. We functionally validated a subset of interactions, including the isoform-specific inhibition of lactate dehydrogenase by long-chain acyl-coenzyme A. Cell treatment with fatty acids caused a loss of pyruvate-lactate interconversion dependent on lactate dehydrogenase isoform expression. These protein-metabolite interactions may contribute to the dynamic, tissue-specific metabolic flexibility that enables growth and survival in an ever-changing nutrient environment.

On the illusion of auxotrophy: met15Δ yeast cells can grow on inorganic sulfur, thanks to the previously uncharacterized homocysteine synthase Yll058w.

Organisms must either synthesize or assimilate essential organic compounds to survive. The homocysteine synthase Met15 has been considered essential for inorganic sulfur assimilation in yeast since its discovery in the 1970s. As a result, MET15 has served as a genetic marker for hundreds of experiments that play a foundational role in eukaryote genetics and systems biology. Nevertheless, we demonstrate here through structural and evolutionary modeling, in vitro kinetic assays, and genetic complementation, that an alternative homocysteine synthase encoded by the previously uncharacterized gene YLL058W enables cells lacking Met15 to assimilate enough inorganic sulfur for survival and proliferation. These cells however fail to grow in patches or liquid cultures unless provided with exogenous methionine or other organosulfurs. We show that this growth failure, which has historically justified the status of MET15 as a classic auxotrophic marker, is largely explained by toxic accumulation of the gas hydrogen sulfide because of a metabolic bottleneck. When patched or cultured with a hydrogen sulfide chelator, and when propagated as colony grids, cells without Met15 assimilate inorganic sulfur and grow, and cells with Met15 achieve even higher yields. Thus, Met15 is not essential for inorganic sulfur assimilation in yeast. Instead, MET15 is the first example of a yeast gene whose loss conditionally prevents growth in a manner that depends on local gas exchange. Our results have broad implications for investigations of sulfur metabolism, including studies of stress response, methionine restriction, and aging. More generally, our findings illustrate how unappreciated experimental variables can obfuscate biological discovery.

Itavastatin and resveratrol increase triosephosphate isomerase protein in a newly identified variant of TPI deficiency.

Triosephosphate isomerase (TPI) deficiency (TPI Df) is an untreatable glycolytic enzymopathy that results in hemolytic anemia, progressive muscular impairment and irreversible brain damage. Although there is a 'common' mutation (TPIE105D), other pathogenic mutations have been described. We identified patients who were compound heterozygous for a newly described mutation, TPIQ181P, and the common TPIE105D mutation. Intriguingly, these patients lacked neuropathy or cognitive impairment. We then initiated biochemical and structural studies of TPIQ181P. Surprisingly, we found that purified TPIQ181P protein had markedly impaired catalytic properties whereas crystallographic studies demonstrated that the TPIQ181P mutation resulted in a highly disordered catalytic lid. We propose that genetic complementation occurs between the two alleles, one with little activity (TPIQ181P) and one with low stability (TPIE105D). Consistent with this, TPIQ181P/E105D fibroblasts exhibit a significant reduction in the TPI protein. These data suggest that impaired stability, and not catalytic activity, is a better predictor of TPI Df severity. Lastly, we tested two recently discovered chemical modulators of mutant TPI stability, itavastatin and resveratrol, and observed a significant increase in TPI in TPIQ181P/E105D patient cells.

Inactivation of RIP3 kinase sensitizes to 15LOX/PEBP1-mediated ferroptotic death.

Ferroptosis and necroptosis are two pro-inflammatory cell death programs contributing to major pathologies and their inhibition has gained attention to treat a wide range of disease states. Necroptosis relies on activation of RIP1 and RIP3 kinases. Ferroptosis is triggered by oxidation of polyunsaturated phosphatidylethanolamines (PUFA-PE) by complexes of 15-Lipoxygenase (15LOX) with phosphatidylethanolamine-binding protein 1 (PEBP1). The latter, also known as RAF kinase inhibitory protein, displays promiscuity towards multiple proteins. In this study we show that RIP3 K51A kinase inactive mice have increased ferroptotic burden and worse outcome after irradiation and brain trauma rescued by anti-ferroptotic compounds Liproxstatin-1 and Ferrostatin 16-86. Given structural homology between RAF and RIP3, we hypothesized that PEBP1 acts as a necroptosis-to-ferroptosis switch interacting with either RIP3 or 15LOX. Using genetic, biochemical, redox lipidomics and computational approaches, we uncovered that PEBP1 complexes with RIP3 and inhibits necroptosis. Elevated expression combined with higher affinity enables 15LOX to pilfer PEBP1 from RIP3, thereby promoting PUFA-PE oxidation and ferroptosis which sensitizes Rip3K51A/K51A kinase-deficient mice to total body irradiation and brain trauma. This newly unearthed PEBP1/15LOX-driven mechanism, along with previously established switch between necroptosis and apoptosis, can serve multiple and diverse cell death regulatory functions across various human disease states.

Resolving the paradox of ferroptotic cell death: Ferrostatin-1 binds to 15LOX/PEBP1 complex, suppresses generation of peroxidized ETE-PE, and protects against ferroptosis.

Hydroperoxy-eicosatetraenoyl-phosphatidylethanolamine (HpETE-PE) is a ferroptotic cell death signal. HpETE-PE is produced by the 15-Lipoxygenase (15LOX)/Phosphatidylethanolamine Binding Protein-1 (PEBP1) complex or via an Fe-catalyzed non-enzymatic radical reaction. Ferrostatin-1 (Fer-1), a common ferroptosis inhibitor, is a lipophilic radical scavenger but a poor 15LOX inhibitor arguing against 15LOX having a role in ferroptosis. In the current work, we demonstrate that Fer-1 does not affect 15LOX alone, however, it effectively inhibits HpETE-PE production by the 15LOX/PEBP1 complex. Computational molecular modeling shows that Fer-1 binds to the 15LOX/PEBP1 complex at three sites and could disrupt the catalytically required allosteric motions of the 15LOX/PEBP1 complex. Using nine ferroptosis cell/tissue models, we show that HpETE-PE is produced by the 15LOX/PEBP1 complex and resolve the long-existing Fer-1 anti-ferroptotic paradox.

Structural and functional divergence of GDAP1 from the glutathione S-transferase superfamily.

Mutations in ganglioside-induced differentiation-associated protein 1 (GDAP1) alter mitochondrial morphology and result in several subtypes of the inherited peripheral neuropathy Charcot-Marie-Tooth disease; however, the mechanism by which GDAP1 functions has remained elusive. GDAP1 contains primary sequence homology to the GST superfamily; however, the question of whether GDAP1 is an active GST has not been clearly resolved. Here, we present biochemical evidence, suggesting that GDAP1 has lost the ability to bind glutathione without a loss of substrate binding activity. We have revealed that the α-loop, located within the H-site motif is the primary determinant for substrate binding. Using structural data of GDAP1, we have found that critical residues and configurations in the G-site which canonically interact with glutathione are altered in GDAP1, rendering it incapable of binding glutathione. Last, we have found that the overexpression of GDAP1 in HeLa cells results in a mitochondrial phenotype which is distinct from oxidative stress-induced mitochondrial fragmentation. This phenotype is dependent on the presence of the transmembrane domain, as well as a unique hydrophobic domain that is not found in canonical GSTs. Together, we data point toward a non-enzymatic role for GDAP1, such as a sensor or receptor.

The Rad51 paralogs facilitate a novel DNA strand specific damage tolerance pathway.

Accurate DNA replication is essential for genomic stability and cancer prevention. Homologous recombination is important for high-fidelity DNA damage tolerance during replication. How the homologous recombination machinery is recruited to replication intermediates is unknown. Here, we provide evidence that a Rad51 paralog-containing complex, the budding yeast Shu complex, directly recognizes and enables tolerance of predominantly lagging strand abasic sites. We show that the Shu complex becomes chromatin associated when cells accumulate abasic sites during S phase. We also demonstrate that purified recombinant Shu complex recognizes an abasic analog on a double-flap substrate, which prevents AP endonuclease activity and endonuclease-induced double-strand break formation. Shu complex DNA binding mutants are sensitive to methyl methanesulfonate, are not chromatin enriched, and exhibit increased mutation rates. We propose a role for the Shu complex in recognizing abasic sites at replication intermediates, where it recruits the homologous recombination machinery to mediate strand specific damage tolerance.

Missense variant in TPI1 (Arg189Gln) causes neurologic deficits through structural changes in the triosephosphate isomerase catalytic site and reduced enzyme levels in vivo.

Mutations in the gene triosephosphate isomerase (TPI) lead to a severe multisystem condition that is characterized by hemolytic anemia, a weakened immune system, and significant neurologic symptoms such as seizures, distal neuropathy, and intellectual disability. No effective therapy is available. Here we report a compound heterozygous patient with a novel TPI pathogenic variant (NM_000365.5:c.569G>A:p.(Arg189Gln)) in combination with the common (NM_000365.5:c.315G>C:p.(Glu104Asp)) allele. We characterized the novel variant by mutating the homologous Arg in Drosophila using a genomic engineering system, demonstrating that missense mutations at this position cause a strong loss of function. Compound heterozygote animals were generated and exhibit motor behavioural deficits and markedly reduced protein levels. Furthermore, examinations of the TPIArg189Gln/TPIGlu104Asp patient fibroblasts confirmed the reduction of TPI levels, suggesting that Arg189Gln may also affect the stability of the protein. The Arg189 residue participates in two salt bridges on the backside of the TPI enzyme dimer, and we reveal that a mutation at this position alters the coordination of the substrate-binding site and important catalytic residues. Collectively, these data reveal a new human pathogenic variant associated with TPI deficiency, identify the Arg189 salt bridge as critical for organizing the catalytic site of the TPI enzyme, and demonstrates that reduced TPI levels are associated with human TPI deficiency. These findings advance our understanding of the molecular pathogenesis of the disease, and suggest new therapeutic avenues for pre-clinical trials.

Characterization of Differential Dynamics, Specificity, and Allostery of Lipoxygenase Family Members.

Accurate modeling of structural dynamics of proteins and their differentiation across different species can help us understand generic mechanisms of function shared by family members and the molecular basis of the specificity of individual members. We focused here on the family of lipoxygenases, enzymes that catalyze lipid oxidation, the mammalian and bacterial structures of which have been elucidated. We present a systematic method of approach for characterizing the sequence, structure, dynamics, and allosteric signaling properties of these enzymes using a combination of structure-based models and methods and bioinformatics tools applied to a data set of 88 structures. The analysis elucidates the signature dynamics of the lipoxygenase family and its differentiation among members, as well as key sites that enable its adaptation to specific substrate binding and allosteric activity.

Empowerment of 15-Lipoxygenase Catalytic Competence in Selective Oxidation of Membrane ETE-PE to Ferroptotic Death Signals, HpETE-PE.

sn2-15-Hydroperoxy-eicasotetraenoyl-phosphatidylethanolamines ( sn2-15-HpETE-PE) generated by mammalian 15-lipoxygenase/phosphatidylethanolamine binding protein-1 (15-LO/PEBP1) complex is a death signal in a recently identified type of programmed cell demise, ferroptosis. How the enzymatic complex selects sn2-ETE-PE as the substrate among 1 of ∼100 total oxidizable membrane PUFA phospholipids is a central, yet unresolved question. To unearth the highly selective and specific mechanisms of catalytic competence, we used a combination of redox lipidomics, mutational and computational structural analysis to show they stem from (i) reactivity toward readily accessible hexagonally organized membrane sn2-ETE-PEs, (ii) relative preponderance of sn2-ETE-PE species vs other sn2-ETE-PLs, and (iii) allosteric modification of the enzyme in the complex with PEBP1. This emphasizes the role of enzymatic vs random stochastic free radical reactions in ferroptotic death signaling.

PEBP1 Wardens Ferroptosis by Enabling Lipoxygenase Generation of Lipid Death Signals.

Ferroptosis is a form of programmed cell death that is pathogenic to several acute and chronic diseases and executed via oxygenation of polyunsaturated phosphatidylethanolamines (PE) by 15-lipoxygenases (15-LO) that normally use free polyunsaturated fatty acids as substrates. Mechanisms of the altered 15-LO substrate specificity are enigmatic. We sought a common ferroptosis regulator for 15LO. We discovered that PEBP1, a scaffold protein inhibitor of protein kinase cascades, complexes with two 15LO isoforms, 15LO1 and 15LO2, and changes their substrate competence to generate hydroperoxy-PE. Inadequate reduction of hydroperoxy-PE due to insufficiency or dysfunction of a selenoperoxidase, GPX4, leads to ferroptosis. We demonstrated the importance of PEBP1-dependent regulatory mechanisms of ferroptotic death in airway epithelial cells in asthma, kidney epithelial cells in renal failure, and cortical and hippocampal neurons in brain trauma. As master regulators of ferroptotic cell death with profound implications for human disease, PEBP1/15LO complexes represent a new target for drug discovery.

Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins.

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are found throughout nature, however, because of their physical properties and tendency to aggregate, they are traditionally viewed as being especially difficult to crystallize. Here, we utilize a variety of quick and simple techniques designed to identify a series of possible domain boundaries for a given coiled-coil protein, and then quickly characterize the behavior of these proteins in solution. With the addition of a strongly fluorescent tag (mRuby2), protein characterization is simple and straightforward. The target protein can be readily visualized under normal lighting and can be quantified with the use of an appropriate imager. The goal is to quickly identify candidates that can be removed from the crystallization pipeline because they are unlikely to succeed, affording more time for the best candidates and fewer funds expended on proteins that do not produce crystals. This process can be iterated to incorporate information gained from initial screening efforts, can be adapted for high-throughput expression and purification procedures, and is augmented by robotic screening for crystallization.

The Histone Modification Domain of Paf1 Complex Subunit Rtf1 Directly Stimulates H2B Ubiquitylation through an Interaction with Rad6.

The five-subunit yeast Paf1 complex (Paf1C) regulates all stages of transcription and is critical for the monoubiquitylation of histone H2B (H2Bub), a modification that broadly influences chromatin structure and eukaryotic transcription. Here, we show that the histone modification domain (HMD) of Paf1C subunit Rtf1 directly interacts with the ubiquitin conjugase Rad6 and stimulates H2Bub independently of transcription. We present the crystal structure of the Rtf1 HMD and use site-specific, in vivo crosslinking to identify a conserved Rad6 interaction surface. Utilizing ChIP-exo analysis, we define the localization patterns of the H2Bub machinery at high resolution and demonstrate the importance of Paf1C in targeting the Rtf1 HMD, and thereby H2Bub, to its appropriate genomic locations. Finally, we observe HMD-dependent stimulation of H2Bub in a transcription-free, reconstituted in vitro system. Taken together, our results argue for an active role for Paf1C in promoting H2Bub and ensuring its proper localization in vivo.

Structure of the Shroom-Rho Kinase Complex Reveals a Binding Interface with Monomeric Shroom That Regulates Cell Morphology and Stimulates Kinase Activity.

Shroom-mediated remodeling of the actomyosin cytoskeleton is a critical driver of cellular shape and tissue morphology that underlies the development of many tissues including the neural tube, eye, intestines, and vasculature. Shroom uses a conserved SD2 domain to direct the subcellular localization of Rho-associated kinase (Rock), which in turn drives changes in the cytoskeleton and cellular morphology through its ability to phosphorylate and activate non-muscle myosin II. Here, we present the structure of the human Shroom-Rock binding module, revealing an unexpected stoichiometry for Shroom in which two Shroom SD2 domains bind independent surfaces on Rock. Mutation of interfacial residues impaired Shroom-Rock binding in vitro and resulted in altered remodeling of the cytoskeleton and loss of Shroom-mediated changes in cellular morphology. Additionally, we provide the first direct evidence that Shroom can function as a Rock activator. These data provide molecular insight into the Shroom-Rock interface and demonstrate that Shroom directly participates in regulating cytoskeletal dynamics, adding to its known role in Rock localization.

Structural and Genetic Studies Demonstrate Neurologic Dysfunction in Triosephosphate Isomerase Deficiency Is Associated with Impaired Synaptic Vesicle Dynamics.

Triosephosphate isomerase (TPI) deficiency is a poorly understood disease characterized by hemolytic anemia, cardiomyopathy, neurologic dysfunction, and early death. TPI deficiency is one of a group of diseases known as glycolytic enzymopathies, but is unique for its severe patient neuropathology and early mortality. The disease is caused by missense mutations and dysfunction in the glycolytic enzyme, TPI. Previous studies have detailed structural and catalytic changes elicited by disease-associated TPI substitutions, and samples of patient erythrocytes have yielded insight into patient hemolytic anemia; however, the neuropathophysiology of this disease remains a mystery. This study combines structural, biochemical, and genetic approaches to demonstrate that perturbations of the TPI dimer interface are sufficient to elicit TPI deficiency neuropathogenesis. The present study demonstrates that neurologic dysfunction resulting from TPI deficiency is characterized by synaptic vesicle dysfunction, and can be attenuated with catalytically inactive TPI. Collectively, our findings are the first to identify, to our knowledge, a functional synaptic defect in TPI deficiency derived from molecular changes in the TPI dimer interface.

Promotion of presynaptic filament assembly by the ensemble of S. cerevisiae Rad51 paralogues with Rad52.

The conserved budding yeast Rad51 paralogues, including Rad55, Rad57, Csm2 and Psy3 are indispensable for homologous recombination (HR)-mediated chromosome damage repair. Rad55 and Rad57 are associated in a heterodimer, while Csm2 and Psy3 form the Shu complex with Shu1 and Shu2. Here we show that Rad55 bridges an interaction between Csm2 with Rad51 and Rad52 and, using a fully reconstituted system, demonstrate that the Shu complex synergizes with Rad55-Rad57 and Rad52 to promote nucleation of Rad51 on single-stranded DNA pre-occupied by replication protein A (RPA). The csm2-F46A allele is unable to interact with Rad55, ablating the ability of the Shu complex to enhance Rad51 presynaptic filament assembly in vitro and impairing HR in vivo. Our results reveal that Rad55-Rad57, the Shu complex and Rad52 act as a functional ensemble to promote Rad51-filament assembly, which has important implications for understanding the role of the human RAD51 paralogues in Fanconi anaemia and cancer predisposition.