Fanconi anemia and Bloom's syndrome crosstalk through FANCJ-BLM helicase interaction

Fanconi anemia (FA) and Bloom's syndrome (BS) are rare hereditary chromosomal instability disorders. FA displays bone marrow failure, acute myeloid leukemia, and head and neck cancers, whereas BS is characterized by growth retardation, immunodeficiency, and a wide spectrum of cancers. The BLM gene mutated in BS encodes a DNA helicase that functions in a protein complex to suppress sister-chromatid exchange. Of the 15 FA genetic complementation groups implicated in interstrand crosslink repair, FANCJ encodes a DNA helicase involved in recombinational repair and replication stress response. Based on evidence that BLM and FANCJ interact we suggest that crosstalk between BLM and FA pathways is more complex than previously thought. We propose testable models for how FANCJ and BLM coordinate to help cells deal with stalled replication forks or double-strand breaks (DSB). Understanding how BLM and FANCJ cooperate will help to elucidate an important pathway for maintaining genomic stability.     

Functional interaction between RNA helicase II/Gu(alpha) and ribosomal protein L4

RNA helicase II/Gu(alpha) is a multifunctional nucleolar protein involved in ribosomal RNA processing in Xenopus laevis oocytes and mammalian cells. Downregulation of Gu(alpha) using small interfering RNA (siRNA) in HeLa cells resulted in 80% inhibition of both 18S and 28S rRNA production. The mechanisms underlying this effect remain unclear. Here we show that in mammalian cells, Gu(alpha) physically interacts with ribosomal protein L4 (RPL4), a component of 60S ribosome large subunit. The ATPase activity of Gu(alpha) is important for this interaction and is also necessary for the function of Gu(alpha) in the production of both 18S and 28S rRNAs. Knocking down RPL4 expression using siRNA in mouse LAP3 cells inhibits the production of 47/45S, 32S, 28S, and 18S rRNAs. This inhibition is reversed by exogenous expression of wild-type human RPL4 protein but not the mutant form lacking Gu(alpha)-interacting motif. These observations have suggested that the function of Gu(alpha) in rRNA processing is at least partially dependent on its ability to interact with RPL4.     

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.     

Role of mammalian RAD51L2 (RAD51C) in recombination and genetic stability

The highly conserved RAD51 protein has a central role in homologous recombination. Five novel RAD51-like genes have been identified in mammalian cells, but little is known about their functions. A DNA damage-sensitive hamster cell line, irs3, was found to have a mutation in the RAD51L2 gene and an undetectable level of RAD51L2 protein. Resistance of irs3 to DNA-damaging agents was significantly increased by expression of the human RAD51L2 gene, but not by other RAD51-like genes or RAD51 itself. Consistent with a role for RAD51L2 in homologous recombination, irs3 cells show a reduction in sister chromatid exchange, an increase in isochromatid breaks, and a decrease in damage-dependent RAD51 focus formation compared with wild type cells. As recently demonstrated for human cells, we show that RAD51L2 forms part of two separate complexes of hamster RAD51-like proteins. Strikingly, neither complex of RAD51-like proteins is formed in irs3 cells. Our results demonstrate that RAD51L2 has a key role in mammalian RAD51-dependent processes, contingent on the formation of protein complexes involved in homologous recombination repair.     

RAD51C (RAD51L2) is involved in maintaining centrosome number in mitosis

The RAD51C (RAD51L2) protein is one out of five RAD51 paralogs and forms a complex that includes either XRCC2 or XRCC3. Both of these complexes may have important functions in homologous recombination (HR). Here, we confirm that the frequency of DNA double-strand break (DSB)-induced HR is reduced in the RAD51C deficient cell line CL-V4B, in agreement with a role for RAD51C in HR. We report that mitotic RAD51C deficient CL-V4B cells also have an increased number of centrosomes in mitosis resulting in aberrant mitotic spindles. These data suggest that the RAD51C protein is important in maintaining correct centrosome numbers and that the complexes including RAD51C and XRCC2 or XRCC3 may be of importance in maintaining correct centrosome numbers in mitosis. Increased centrosome numbers following a RAD51C defect indicates that this protein might be important in preventing aneuploidy, suggesting that it could be a potential tumour suppressor in mammals.     

Potential role for the BLM helicase in recombinational repair via a conserved interaction with RAD51

Bloom's syndrome (BS) is an autosomal recessive disorder that predisposes individuals to a wide range of cancers. The gene mutated in BS, BLM, encodes a member of the RecQ family of DNA helicases. The precise role played by these enzymes in the cell remains to be determined. However, genome-wide hyper-recombination is a feature of many RecQ helicase-deficient cells. In eukaryotes, a central step in homologous recombination is catalyzed by the RAD51 protein. In response to agents that induce DNA double-strand breaks, RAD51 accumulates in nuclear foci that are thought to correspond to sites of recombinational repair. Here, we report that purified BLM and human RAD51 interact in vitro and in vivo, and that residues in the N- and C-terminal domains of BLM can independently mediate this interaction. Consistent with these observations, BLM localizes to a subset of RAD51 nuclear foci in normal human cells. Moreover, the number of BLM foci and the extent to which BLM and RAD51 foci co-localize increase in response to ionizing radiation. Nevertheless, the formation of RAD51 foci does not require functional BLM. Indeed, in untreated BS cells, an abnormally high proportion of the cells contain RAD51 nuclear foci. Exogenous expression of BLM markedly reduces the fraction of cells containing RAD51 foci. The interaction between BLM and RAD51 appears to have been evolutionarily conserved since the C-terminal domain of Sgs1, the Saccharomyces cerevisiae homologue of BLM, interacts with yeast Rad51. Furthermore, genetic analysis reveals that the SGS1 and RAD51 genes are epistatic indicating that they operate in a common pathway. Potential roles for BLM in the RAD51 recombinational repair pathway are discussed.     

The interaction profile of homologous recombination repair proteins RAD51C,RAD51D and XRCC2 as determined by proteomic analysis

The RAD51 family of proteins is involved in homologous recombination (HR) DNA repair and maintaining chromosome integrity. To identify candidates that interact with HR proteins, the mouse RAD51C, RAD51D and XRCC2 proteins were purified using bacterial expression systems and each of them used to co‐precipitate interacting partners from mouse embryonic fibroblast cellular extracts. Mass spectroscopic analysis was performed on protein bands obtained after 1‐D SDS‐PAGE of co‐precipitation eluates from cell extracts of mitomycin C treated and untreated mouse embryonic fibroblasts. Profiling of the interacting proteins showed a clear bias toward nucleic acid binding and modification proteins. Interactions of four candidate proteins (SFPQ, NONO, MSH2 and mini chromosome maintenance protein 2) were confirmed by Western blot analysis of co‐precipitation eluates and were also verified to form ex vivo complexes with RAD51D. Additional interacting proteins were associated with cell division, embryo development, protein and carbohydrate metabolism, cellular trafficking, protein synthesis, modification or folding, and cell structure or motility functions. Results from this study are an important step toward identifying interacting partners of the RAD51 paralogs and understanding the functional diversity of proteins that assist or regulate HR repair mechanisms.     

Identification of functional domains in the RAD51L2 (RAD51C) protein and its requirement for gene conversion

The RAD51 protein plays a key part in the process of homologous recombination through its catalysis of homologous DNA pairing and strand exchange. Additionally five novel mammalian RAD51-like proteins have been identified in mammalian cells, but their roles in homologous recombination are much less well established. These RAD51-like proteins form two different complexes, but only the RAD51L2 (RAD51C) protein is a part of both complexes. By using site-directed mutagenesis of RAD51L2, we show that non-conservative mutation of the putative ATP-binding domain severely reduces its function, whereas a conservative mutation shows partial loss of function. We find that the protein is localized to the nucleus by tagging RAD51L2 with the green fluorescent protein and provisionally identify a C-terminal domain that acts as a nuclear localization signal. Further, a RAD51L2-deficient cell line was found to have significantly reduced homology-directed repair of a DNA double-strand break by gene conversion. This recombination defect could be partially restored by ectopic expression of the human RAD51L2 protein. Therefore we have identified protein domains that are important for the correct functioning of RAD51L2 and have shown that there is a specific requirement for RAD51L2 in gene conversion in mammalian cells.     

The RAD51 family member, RAD51L3, is a DNA-stimulated ATPase that forms a complex with XRCC2

The Rad51 protein in eukaryotic cells is a structural and functional homolog of Escherichia coli RecA with a role in DNA repair and genetic recombination. Several proteins showing sequence similarity to Rad51 have previously been identified in both yeast and human cells. In Saccharomyces cerevisiae, two of these proteins, Rad55p and Rad57p, form a heterodimer that can stimulate Rad51-mediated DNA strand exchange. Here, we report the purification of one of the representatives of the RAD51 family in human cells. We demonstrate that the purified RAD51L3 protein possesses single-stranded DNA binding activity and DNA-stimulated ATPase activity, consistent with the presence of "Walker box" motifs in the deduced RAD51L3 sequence. We have identified a protein complex in human cells containing RAD51L3 and a second RAD51 family member, XRCC2. By using purified proteins, we demonstrate that the interaction between RAD51L3 and XRCC2 is direct. Given the requirements for XRCC2 in genetic recombination and protection against DNA-damaging agents, we suggest that the complex of RAD51L3 and XRCC2 is likely to be important for these functions in human cells.     

Functional link between BLM defective in Bloom's syndrome and the ataxia-telangiectasia-mutated protein,ATM

Chromosome aberrations, genomic instability, and cancer predisposition are hallmarks of a number of syndromes in which the defective genes recognize and/or repair DNA damage or are involved in some aspect of DNA processing. We report here direct interaction between BLM, mutated in Bloom's Syndrome (BS), and ATM, mutated is ataxia-telangiectasia, and we have mapped the sites of interaction. Full-length BLM cDNA corrected sister chromatid exchange (SCE) and radiosensitivity in BS cells. Mitotic phosphorylation of BLM was partially dependent on ATM, and phosphorylation sites on BLM were identified. A phosphospecific antibody against one of these sites (Thr-99) revealed radiation-induced phosphorylation, which was defective in ataxia-telangiectasia cells. Stable cell lines expressing phosphorylation site mutants failed to correct radiosensitivity in BS cells but corrected SCE. These mutants also sensitized normal control cells to radiation and increased radiation-induced chromosome aberrations but did not cause SCE numbers to increase. These data suggest that ATM and BLM function together in recognizing abnormal DNA structures by direct interaction and that these phosphorylation sites in BLM are important for radiosensitivity status but not for SCE frequency.     

Strand annealing and motor driven activities of SMARCAL1 and ZRANB3 are stimulated by RAD51 and the paralog complex

SMARCAL1, ZRANB3 and HLTF are required for the remodeling of replication forks upon stress to promote genome stability. RAD51, along with the RAD51 paralog complex, were also found to have recombination-independent functions in fork reversal, yet the underlying mechanisms remained unclear. Using reconstituted reactions, we build upon previous data to show that SMARCAL1, ZRANB3 and HLTF have unequal biochemical capacities, explaining why they have non-redundant functions. SMARCAL1 uniquely anneals RPA-coated ssDNA, which depends on its direct interaction with RPA, but not on ATP. SMARCAL1, along with ZRANB3, but not HLTF efficiently employ ATPase driven translocase activity to rezip RPA-covered bubbled DNA, which was proposed to mimic elements of fork reversal. In contrast, ZRANB3 and HLTF but not SMARCAL1 are efficient in branch migration that occurs downstream in fork remodeling. We also show that low concentrations of RAD51 and the RAD51 paralog complex, RAD51B–RAD51C–RAD51D–XRCC2 (BCDX2), directly stimulate the motor-driven activities of SMARCAL1 and ZRANB3 but not HLTF, and the interplay is underpinned by physical interactions. Our data provide a possible mechanism explaining previous cellular experiments implicating RAD51 and BCDX2 in fork reversal.     

DNA strand displacement, strand annealing and strand swapping by the Drosophila Bloom's syndrome helicase

Genetic analysis of the Drosophila Bloom's syndrome helicase homolog (mus309/DmBLM) indicates that DmBLM is required for the synthesis-dependent strand annealing (SDSA) pathway of homologous recombination. Here we report the first biochemical study of DmBLM. Recombinant, epitope-tagged DmBLM was expressed in Drosophila cell culture and highly purified protein was prepared from nuclear extracts. Purified DmBLM exists exclusively as a high molecular weight (~1.17 MDa) species, is a DNA-dependent ATPase, has 3′→5′ DNA helicase activity, prefers forked substrate DNAs and anneals complementary DNAs. High-affinity DNA binding is ATP-dependent and low-affinity ATP-independent interactions contribute to forked substrate DNA binding and drive strand annealing. DmBLM combines DNA strand displacement with DNA strand annealing to catalyze the displacement of one DNA strand while annealing a second complementary DNA strand.     

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.     

Functional and physical interaction between Bcl-X(L) and a BH3-like domain in Beclin-1

The anti-apoptotic proteins Bcl-2 and Bcl-X(L) bind and inhibit Beclin-1, an essential mediator of autophagy. Here, we demonstrate that this interaction involves a BH3 domain within Beclin-1 (residues 114-123). The physical interaction between Beclin-1 and Bcl-X(L) is lost when the BH3 domain of Beclin-1 or the BH3 receptor domain of Bcl-X(L) is mutated. Mutation of the BH3 domain of Beclin-1 or of the BH3 receptor domain of Bcl-X(L) abolishes the Bcl-X(L)-mediated inhibition of autophagy triggered by Beclin-1. The pharmacological BH3 mimetic ABT737 competitively inhibits the interaction between Beclin-1 and Bcl-2/Bcl-X(L), antagonizes autophagy inhibition by Bcl-2/Bcl-X(L) and hence stimulates autophagy. Knockout or knockdown of the BH3-only protein Bad reduces starvation-induced autophagy, whereas Bad overexpression induces autophagy in human cells. Gain-of-function mutation of the sole BH3-only protein from Caenorhabditis elegans, EGL-1, induces autophagy, while deletion of EGL-1 compromises starvation-induced autophagy. These results reveal a novel autophagy-stimulatory function of BH3-only proteins beyond their established role as apoptosis inducers. BH3-only proteins and pharmacological BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin-1 and Bcl-2 or Bcl-X(L).     

The Bloom's syndrome helicase (BLM) interacts physically and functionally with p12, the smallest subunit of human DNA polymerase delta

Bloom's syndrome (BS) is a cancer predisposition disorder caused by mutation of the BLM gene, encoding a member of the RecQ helicase family. Although the phenotype of BS cells is suggestive of a role for BLM in repair of stalled or damaged replication forks, thus far there has been no direct evidence that BLM associates with any of the three human replicative DNA polymerases. Here, we show that BLM interacts specifically in vitro and in vivo with p12, the smallest subunit of human POL δ (hPOL δ). The hPOL δ enzyme, as well as the isolated p12 subunit, stimulates the DNA helicase activity of BLM. Conversely, BLM stimulates hPOL δ strand displacement activity. Our results provide the first functional link between BLM and the replicative machinery in human cells, and suggest that BLM might be recruited to sites of disrupted replication through an interaction with hPOL δ. Finally, our data also define a novel role for the poorly characterized p12 subunit of hPOL δ.     

Phosphorylation of the Bloom's syndrome helicase and its role in recovery from S-phase arrest

Bloom's syndrome (BS) is a human genetic disorder associated with cancer predisposition. The BS gene product, BLM, is a member of the RecQ helicase family, which is required for the maintenance of genome stability in all organisms. In budding and fission yeasts, loss of RecQ helicase function confers sensitivity to inhibitors of DNA replication, such as hydroxyurea (HU), by failure to execute normal cell cycle progression following recovery from such an S-phase arrest. We have examined the role of the human BLM protein in recovery from S-phase arrest mediated by HU and have probed whether the stress-activated ATR kinase, which functions in checkpoint signaling during S-phase arrest, plays a role in the regulation of BLM function. We show that, consistent with a role for BLM in protection of human cells against the toxicity associated with arrest of DNA replication, BS cells are hypersensitive to HU. BLM physically associates with ATR (ataxia telangiectasia and rad3(+) related) protein and is phosphorylated on two residues in the N-terminal domain, Thr-99 and Thr-122, by this kinase. Moreover, BS cells ectopically expressing a BLM protein containing phosphorylation-resistant T99A/T122A substitutions fail to adequately recover from an HU-induced replication blockade, and the cells subsequently arrest at a caffeine-sensitive G(2)/M checkpoint. These abnormalities are not associated with a failure of the BLM-T99A/T122A protein to localize to replication foci or to colocalize either with ATR itself or with other proteins that are required for response to DNA damage, such as phosphorylated histone H2AX and RAD51. Our data indicate that RecQ helicases play a conserved role in recovery from perturbations in DNA replication and are consistent with a model in which RecQ helicases act to restore productive DNA replication following S-phase arrest and hence prevent subsequent genomic instability.