Biochemical characterization of the human RAD51 protein. I. ATP hydrolysis

The prototypical bacterial RecA protein promotes recombination/repair by catalyzing strand exchange between homologous DNAs. While the mechanism of strand exchange remains enigmatic, ATP-induced cooperativity between RecA protomers is critical for its function. A human RecA homolog, human RAD51 protein (hRAD51), facilitates eukaryotic recombination/repair, although its ability to hydrolyze ATP and/or promote strand exchange appears distinct from the bacterial RecA. We have quantitatively examined the hRAD51 ATPase. The catalytic efficiency (k(cat)/K(m)) of the hRAD51 ATPase was approximately 50-fold lower than the RecA ATPase. Altering the ratio of DNA/hRAD51 and including salts that stimulate DNA strand exchange (ammonium sulfate and spermidine) were found to affect the catalytic efficiency of hRAD51. The average site size of hRAD51 was determined to be approximately 3 nt (bp) for both single-stranded and double-stranded DNA. Importantly, hRAD51 lacks the magnitude of ATP-induced cooperativity that is a hallmark of RecA. Together, these results suggest that hRAD51 may be unable to coordinate ATP hydrolysis between neighboring protomers.     

Biochemical characterization of the human RAD51 protein. III. Modulation of DNA binding by adenosine nucleotides

Adenosine nucleotides affect the ability of RecA small middle dotsingle-stranded DNA (ssDNA) nucleoprotein filaments to cooperatively assume and maintain an extended structure that facilitates DNA pairing during recombination. Here we have determined that ADP and ATP/ATPgammaS affect the DNA binding and aggregation properties of the human RecA homolog human RAD51 protein (hRAD51). These studies have revealed significant differences between hRAD51 and RecA. In the presence of ATPgammaS, RecA forms a stable complex with ssDNA, while the hRAD51 ssDNA complex is destabilized. Conversely, in the presence of ADP and ATP, the RecA ssDNA complex is unstable, while the hRAD51 ssDNA complex is stabilized. We identified two hRAD51 small middle dotssDNA binding forms by gel shift analysis, which were distinct from a well defined RecA small middle dotssDNA binding form. The available evidence suggests that a low molecular weight hRAD51 small middle dotssDNA binding form (hRAD51 small middle dotssDNA(low)) correlates with active ADP and ATP processing. A high molecular weight hRAD51 small middle dotssDNA aggregate (hRAD51 small middle dotssDNA(high)) appears to correlate with a form that fails to process ADP and ATP. Our data are consistent with the notion that hRAD51 is unable to appropriately coordinate ssDNA binding with adenosine nucleotide processing. These observations suggest that other factors may assist hRAD51 in order to mirror RecA recombinational function.     

Conformational changes modulate the activity of human RAD51 protein

Homologous recombination provides a major pathway for the repair of DNA double-strand breaks in mammalian cells. Defects in homologous recombination can lead to high levels of chromosomal translocations or deletions, which may promote cell transformation and cancer development. A key component of this process is RAD51. In comparison to RecA, the bacterial homologue, human RAD51 protein exhibits low-level strand-exchange activity in vitro. This activity can, however, be stimulated by the presence of high salt. Here, we have investigated the mechanistic basis for this stimulation. We show that high ionic strength favours the co-aggregation of RAD51-single-stranded DNA (ssDNA) nucleoprotein filaments with naked duplex DNA, to form a complex in which the search for homologous sequences takes place. High ionic strength allows differential binding of RAD51 to ssDNA and double-stranded DNA (dsDNA), such that ssDNA-RAD51 interactions are unaffected, whereas those between RAD51 and dsDNA are destabilized. Most importantly, high salt induces a conformational change in RAD51, leading to the formation of extended nucleoprotein filaments on ssDNA. These extended filaments mimic the active form of the Escherichia coli RecA-ssDNA filament that exhibits efficient strand-exchange activity.     

RAD51C interacts with RAD51B and is central to a larger protein complex in vivo exclusive of RAD51.

RAD51B and RAD51C are two of five known paralogs of the human RAD51 protein that are thought to function in both homologous recombination and DNA double-strand break repair. This work describes the in vitro and in vivo identification of the RAD51B/RAD51C heterocomplex. The RAD51B/RAD51C heterocomplex was isolated and purified by immunoaffinity chromatography from insect cells co-expressing the recombinant proteins. Moreover, co-immunoprecipitation of the RAD51B and RAD51C proteins from HeLa, MCF10A, and MCF7 cells strongly suggests the existence of an endogenous RAD51B/RAD51C heterocomplex. We extended these observations to examine the interaction between the RAD51B/RAD51C complex and the other RAD51 paralogs. Immunoprecipitation using protein-specific antibodies showed that RAD51C is central to a single large protein complex and/or several smaller complexes with RAD51B, RAD51D, XRCC2, and XRCC3. However, our experiments showed no evidence for the inclusion of RAD51 within these complexes. Further analysis is required to elucidate the function of the RAD51B/RAD51C heterocomplex and its association with the other RAD51 paralogs in the processes of homologous recombination and DNA double-strand break repair.     

XRCC2 is a nuclear RAD51-like protein required for damage-dependent RAD51 focus formation without the need for ATP binding

The human XRCC2 gene was recently identified by its ability to complement a hamster cell line, irs1, which is sensitive to DNA-damaging agents and shows genetic instability. The XRCC2 protein is highly conserved in mammalian species and has structural features, including a putative ATP-binding domain (P-loop), consistent with membership of the RecA/RAD51 family of recombination-repair proteins. We show that a hybrid XRCC2-green fluorescent protein, which was found to be functional by complementation, localizes to the nucleus. We have established a functional link between XRCC2 and RAD51 by looking at damage-dependent RAD51 focus formation in the irs1 cell line. Little or no formation of RAD51 foci occurred in irs1. This effect was specific to the loss of XRCC2 because transfection of the gene into irs1 restored normal levels of focus formation. Surprisingly, XRCC2 genes carrying site-specific mutations in P-loop residues were found to be able to complement the XRCC2-deficient irs1 line for a number of different end points. We conclude that XRCC2 is important in the early stages of homologous recombination in mammalian cells to facilitate RAD51-dependent recombination repair but that it does not make use of ATP binding to promote this function.     

RAD51AP1 is a structure-specific DNA binding protein that stimulates joint molecule formation during RAD51-mediated homologous recombination

Homologous recombination is essential for preserving genome integrity. Joining of homologous DNA molecules through strand exchange, a pivotal step in recombination, is mediated by RAD51. Here, we identify RAD51AP1 as a RAD51 accessory protein that specifically stimulates joint molecule formation through the combination of structure-specific DNA binding and physical contact with RAD51. At the cellular level, we show that RAD51AP1 is required to protect cells from the adverse effects of DNA double-strand break-inducing agents. At the biochemical level, we show that RAD51AP1 has a selective affinity for branched-DNA structures that are obligatory intermediates during joint molecule formation. Our results highlight the importance of structural transitions in DNA as control points in recombination. The affinity of RAD51AP1 for the central protein and DNA intermediates of recombination confers on it the ability to control the preservation of genome integrity at a number of critical mechanistic steps.     

Identification and characterization of the RAD51 gene from the ciliate Tetrahymena thermophila.

The RAD51 gene is a eukaryotic homolog of rec A, a critical component in homologous recombination and DNA repair pathways in Escherichia coli . We have cloned the RAD51 homolog from Tetrahymena thermophila , a ciliated protozoan. Tetrahymena thermophila RAD51 encodes a 36.3 kDa protein whose amino acid sequence is highly similar to representative Rad51 homologs from other eukaryotic taxa. Recombinant Rad51 protein was purified to near homogeneity following overproduction in a bacterial expression system. The purified protein binds to both single- and double-stranded DNA, possesses a DNA-dependent ATPase activity and promotes intermolecular ligation of linearized plasmid DNA. While steady-state levels of Rad51 mRNA are low in normally growing cells, treatment with UV light resulted in a >100-fold increase in mRNA levels. This increase in mRNA was time dependent, but relatively independent of UV dose over a range of 1400-5200 J/m2. Western blot analysis confirmed that Rad51 protein levels increase upon UV irradiation. Exposure to the alkylating agent methyl methane sulfonate also resulted in substantially elevated Rad51 protein levels in treated cells, with pronounced localization in the macronucleus. These data are consistent with the hypothesis that ciliates such as T.thermophila utilize a Rad51-dependent pathway to repair damaged DNA.     

Sequence, chromosomal location and expression analysis of the murine homologue of human RAD51L2/RAD51C.

The Rad51 protein has been shown to play a vital role in the DNA repair process. In humans, its interaction with proteins like BRCA1 and BRCA2 has provided an insight into the mechanism of how these molecules function as tumor suppressors. Several members of the Rad51-like family have been recently identified, including RAD51L2. This gene has been found to be amplified in breast tumors suggesting its role in tumor progression. Here, we describe the cloning of the murine homologue of the human RAD51L2/RAD51C gene. Sequence analysis has revealed that the murine Rad51l2 protein is 86% identical and 93% similar to its human homologue. In spite of such high sequence conservation, the murine protein lacks the first nine amino acids present in the human protein. We have cloned and confirmed the sequence of the 5' end of the murine Rad51l2 cDNA using 5' RACE technique as well as by sequencing the genomic region flanking the first exon of the murine Rad51l2 gene. Northern analysis shows that Rad51l2 is expressed in several adult tissues as well as in embryos at various developmental stages. The murine Rad51l2 gene maps to chromosome 11 and is located in the syntenic region of human chromosome 17q22-23, where the human RAD51L2 is present.     

Roles of XRCC2, RAD51B and RAD51D in RAD51-Independent SSA Recombination

The repair of DNA double-strand breaks by recombination is key to the maintenance of genome integrity in all living organisms. Recombination can however generate mutations and chromosomal rearrangements, making the regulation and the choice of specific pathways of great importance. In addition to end-joining through non-homologous recombination pathways, DNA breaks are repaired by two homology-dependent pathways that can be distinguished by their dependence or not on strand invasion catalysed by the RAD51 recombinase. Working with the plant Arabidopsis thaliana, we present here an unexpected role in recombination for the Arabidopsis RAD51 paralogues XRCC2, RAD51B and RAD51D in the RAD51-independent single-strand annealing pathway. The roles of these proteins are seen in spontaneous and in DSB-induced recombination at a tandem direct repeat recombination tester locus, both of which are unaffected by the absence of RAD51. Individual roles of these proteins are suggested by the strikingly different severities of the phenotypes of the individual mutants, with the xrcc2 mutant being the most affected, and this is confirmed by epistasis analyses using multiple knockouts. Notwithstanding their clearly established importance for RAD51-dependent homologous recombination, XRCC2, RAD51B and RAD51D thus also participate in Single-Strand Annealing recombination.     

Biochemical characterization of human S-nitrosohemoglobin. Effects on oxygen binding and transnitrosation.

S-Nitrosation of cysteine beta93 in hemoglobin (S-nitrosohemoglobin (SNO-Hb)) occurs in vivo, and transnitrosation reactions of deoxygenated SNO-Hb are proposed as a mechanism leading to release of NO and control of blood flow. However, little is known of the oxygen binding properties of SNO-Hb or the effects of oxygen on transnitrosation between SNO-Hb and the dominant low molecular weight thiol in the red blood cell, GSH. These data are important as they would provide a biochemical framework to assess the physiological function of SNO-Hb. Our results demonstrate that SNO-Hb has a higher affinity for oxygen than native Hb. This implies that NO transfer from SNO-Hb in vivo would be limited to regions of extremely low oxygen tension if this were to occur from deoxygenated SNO-Hb. Furthermore, the kinetics of the transnitrosation reactions between GSH and SNO-Hb are relatively slow, making transfer of NO+ from SNO-Hb to GSH less likely as a mechanism to elicit vessel relaxation under conditions of low oxygen tension and over the circulatory lifetime of a given red blood cell. These data suggest that the reported oxygen-dependent promotion of S-nitrosation from SNO-Hb involves biochemical mechanisms that are not intrinsic to the Hb molecule.     

Biochemical characterization of the tetrodotoxin binding protein from Electrophorus electricus

Biochemical properties of a detergent-solubilized tetrodotoxin binding component from Electrophorus electricus have been examined and compared with those found for the membrane-bound protein. The toxin binding component was solubilized with high efficiency by a variety of nonionic detergents and with lower efficiency by sodium cholate and deoxycholate. Detergent-solubilized preparations bound tetrodotoxin and saxitoxin tightly and specifically, and this binding was observed to be rapidly and irreversibly blocked by carboxylate-modifying reagents. Inactivation by carbodiimide and glycine ester or by a trimethyloxonium salt could be prevented by tetrodotoxin occupancy of the binding site. Tetrodotoxin binding activity in both solubilized preparations and in membranes was found to be highly resistant to proteases. In contrast, the activity was extremely sensitive to the action of phospholipase A2. The biochemical properties of the tetrodotoxin binding component solubilized in mixed lipid-detergent micelles are similar to those found in native membranes, with respect to the characteristics of equilibrium toxin binding and to the sensitivity of toxin binding activity to chemical modification and degradative enzymes. There were some differences with respect to the kinetics of tetrodotoxin binding. In addition, the tetrodotoxin binding component from eel is shown to behave as a glycoprotein, being selectively absorbed to resins coupled to concanavalin A, wheat germ agglutinin, Lens culinaris lectin, and ricin with the appropriate glycoside.     

Visualization of direct and diffusion-assisted RAD51 nucleation by full-length human BRCA2 protein

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Homologous Pairing Activities of Two Rice RAD51 Proteins,RAD51A1 and RAD51A2

In higher eukaryotes, RAD51 functions as an essential protein in homologous recombination and recombinational repair of DNA double strand breaks. During these processes, RAD51 catalyzes homologous pairing between single-stranded DNA and double-stranded DNA. Japonica cultivars of rice (Oryza sativa) encode two RAD51 proteins, RAD51A1 and RAD51A2, whereas only one RAD51 exists in yeast and mammals. However, the functional differences between RAD51A1 and RAD51A2 have not been elucidated, because their biochemical properties have not been characterized. In the present study, we purified RAD51A1 and RAD51A2, and found that RAD51A2 robustly promotes homologous pairing in vitro. RAD51A1 also possesses homologous-pairing activity, but it is only about 10% of the RAD51A2 activity. Both RAD51A1 and RAD51A2 bind to ssDNA and dsDNA, and their DNA binding strictly requires ATP, which modulates the polymer formation activities of RAD51A1 and RAD51A2. These findings suggest that although both RAD51A1 and RAD51A2 have the potential to catalyze homologous pairing, RAD51A2 may be the major recombinase in rice.     

Biochemical characterization of human dynamin-like protein 1

Human dynamin-like protein 1 (DLP-1) is involved in the fission of mitochondrial outer membranes, a process that helps to maintain mitochondrial morphology and to reduce the accumulation of functional and structural defects in mitochondria. DLP-1 is a ~80 kDa membrane-interacting protein and contains a GTPase domain, a middle domain, a putative PH-like domain and a GTPase effector domain (GED). While the GED has been suggested to be important on protein oligomerization and GTPase activation, functional relationships between the other domains especially the roles of the middle domain in protein activity remains less clear. In this study, we have investigated the biochemical properties of recombinant DLP-1 wild-type and selected mutants, all expressed in Escherichia coli. The middle domain mutants G350D, R365S and ΔPH (lacking the putative PH-like domain) severely impair the GTPase activity, but have no obvious effects on protein tetramerization and liposome-binding properties, suggesting these mutants probably affect protein intra-molecular interactions. Our study also suggested that proper domain-domain interactions are important for DLP-1 GTPase activity.     

Functional interaction between the Bloom's syndrome helicase and the RAD51 paralog,RAD51L3 (RAD51D)

Bloom's syndrome (BS) is a genetic disorder associated with short stature, fertility defects, and a predisposition to the development of cancer. BS cells are characterized by genomic instability; in particular, a high rate of reciprocal exchanges between sister-chromatids and homologous chromosomes. The BS gene product, BLM, is a helicase belonging to the highly conserved RecQ family. BLM is known to form a complex with the RAD51 recombinase, and to act upon DNA intermediates that form during homologous recombination, including D-loops and Holliday junctions. Here, we show that BLM also makes a direct physical association with the RAD51L3 protein (also known as RAD51D), a so-called RAD51 paralog that shows limited sequence similarity to RAD51 itself. This interaction is mediated through the N-terminal domain of BLM. To analyze functional interactions between BLM and RAD51L3, we have purified a heteromeric complex comprising RAD51L3 and a second RAD51 paralog, XRCC2. We show that the RAD51L3-XRCC2 complex stimulates BLM to disrupt synthetic 4-way junctions that model the Holliday junction. We also show that a truncated form of BLM, which retains helicase activity but is unable to bind RAD51L3, is not stimulated by the RAD51L3-XRCC2 complex. Our data indicate that the activity of BLM is modulated through an interaction with the RAD51L3-XRCC2 complex, and that this stimulatory effect on BLM is dependent upon a direct physical association between the BLM and RAD51L3 proteins. We propose that BLM co-operates with RAD51 paralogs during the late stages of homologous recombination processes that serve to restore productive DNA replication at sites of damaged or stalled replication forks.     

Defining the salt effect on human RAD51 activities

Previous work by Sung and colleagues identified unusual salt requirements for hRAD51 strand exchange compared to RecA [S. Sigurdsson, K. Trujillo, B. Song, S. Stratton, P. Sung, Basis for avid homologous DNA strand exchange by human Rad51 and RPA, J. Biol. Chem. 276 (2001) 8798-8806]. Later studies showed that this salt [(NH4)2SO4] appeared to enhance the ability of hRAD51 to distinguish ssDNA from dsDNA [Y. Liu, A.Z. Stasiak, J.Y. Masson, M.J. McIlwraith, A. Stasiak, S.C. West, Conformational changes modulate the activity of human RAD51 protein, J. Mol. Biol. 337 (2004) 817-827]. The mechanism of this salt effect remains enigmatic. Here, we detail the properties of several neutral salts on hRAD51 activities. We found that the cation identity correlated with the stimulatory effect of these neutral salts on hRAD51 ATPase and strand exchange activities. The salt effect appears to be related to the size of the cation, which may be largely mimicked with the cesium ion. These results are consistent with the hypothesis that stimulating cations induce an important conformation and/or transition state in hRAD51. In the presence of an optimal ammonium-based salt (NaNH4HPO4), hRAD51 mediated strand exchange was successfully performed using a simplified protocol. We confirmed and extend the observation that efficient strand exchange correlated with preferential binding of ssDNA over dsDNA. In addition we observed an induced stability of the hRAD51-DNA complex in the presence of ATP that becomes unstable following ATP hydrolysis (the ADP form or nucleotide free form). These salt-induced characteristics of hRAD51 increasingly resemble RecA-mediated recombinase activities, which should help in dissecting the mechanism of these proteins in homologous recombination.     

Precise binding of single-stranded DNA termini by human RAD52 protein

The human RAD52 protein, which exhibits a heptameric ring structure, has been shown to bind resected double strand breaks (DSBs), consistent with an early role in meiotic recombination and DSB repair. In this work, we show that RAD52 binds single-stranded and tailed duplex DNA molecules via precise interactions with the terminal base. When probed with hydroxyl radicals, ssDNA-RAD52 complexes exhibit a four-nucleotide repeat hypersensitivity pattern. This unique pattern is due to the interaction of RAD52 with either a 5' or a 3' terminus of the ssDNA, is sequence independent and is phased precisely from the terminal nucleotide. Hypersensitivity is observed over approximately 36 nucleotides, consistent with the length of DNA that is protected by RAD52 in nuclease protection assays. We propose that RAD52 binds DNA breaks via specific interactions with the terminal base, leading to the formation of a precisely organized ssDNA-RAD52 complex in which the DNA lies on an exposed surface of the protein. This protein-DNA arrangement may facilitate the DNA-DNA interactions necessary for RAD52-mediated annealing of complementary DNA strands.     

Defects in recombination activity caused by somatic and germline mutations in the multimerization/BRCA2 binding region of human RAD51 protein

The human RAD51 recombinase possesses DNA pairing and strand exchange activities that are essential for the error-free, homology-directed repair of DNA double-strand breaks. The recombination activities of RAD51 are activated upon its assembly into presynaptic filaments on single-stranded DNA at resected DSB ends. Defects in filament assembly caused by mutations in RAD51 or its regulators such as BRCA2 are associated with human cancer. Here we describe two novel RAD51 missense variants located in the multimerization/BRCA2 binding region of RAD51. F86L is a breast tumor-derived somatic variant that affects the interface between adjacent RAD51 protomers in the presynaptic filament. E258A is a germline variant that maps to the interface region between the N-terminal and RecA homology domains of RAD51. Both variants exhibit abnormal biochemistry including altered DNA strand exchange activity. Both variants inhibit the DNA strand exchange activity of wild-type RAD51, suggesting a mechanism for negative dominance. The inhibitory effect of F86L on wild-type RAD51 is surprising since F86L alone exhibits robust DNA strand exchange activity. Our findings indicate that even DNA strand exchange-proficient variants can have negative functional interactions with wild-type RAD51. Thus heterozygous F86L or E258 mutations in RAD51 could promote genomic instability, and thereby contribute to tumor progression.