Sequential role of RAD51 paralog complexes in replication fork remodeling and restart.

Homologous recombination (HR) factors were recently implicated in DNA replication fork remodeling and protection. While maintaining genome stability, HR-mediated fork remodeling promotes cancer chemoresistance, by as-yet elusive mechanisms. Five HR cofactors - the RAD51 paralogs RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3 - recently emerged as crucial tumor suppressors. Albeit extensively characterized in DNA repair, their role in replication has not been addressed systematically. Here, we identify all RAD51 paralogs while screening for modulators of RAD51 recombinase upon replication stress. Single-molecule analysis of fork progression and architecture in isogenic cellular systems shows that the BCDX2 subcomplex restrains fork progression upon stress, promoting fork reversal. Accordingly, BCDX2 primes unscheduled degradation of reversed forks in BRCA2-defective cells, boosting genomic instability. Conversely, the CX3 subcomplex is dispensable for fork reversal, but mediates efficient restart of reversed forks. We propose that RAD51 paralogs sequentially orchestrate clinically relevant transactions at replication forks, cooperatively promoting fork remodeling and restart.

Differential Requirements for the RAD51 Paralogs in Genome Repair and Maintenance in Human Cells.

Deficiency in several of the classical human RAD51 paralogs [RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3] is associated with cancer predisposition and Fanconi anemia. To investigate their functions, isogenic disruption mutants for each were generated in non-transformed MCF10A mammary epithelial cells and in transformed U2OS and HEK293 cells. In U2OS and HEK293 cells, viable ablated clones were readily isolated for each RAD51 paralog; in contrast, with the exception of RAD51B, RAD51 paralogs are cell-essential in MCF10A cells. Underlining their importance for genomic stability, mutant cell lines display variable growth defects, impaired sister chromatid recombination, reduced levels of stable RAD51 nuclear foci, and hyper-sensitivity to mitomycin C and olaparib, with the weakest phenotypes observed in RAD51B-deficient cells. Altogether these observations underscore the contributions of RAD51 paralogs in diverse DNA repair processes, and demonstrate essential differences in different cell types. Finally, this study will provide useful reagents to analyze patient-derived mutations and to investigate mechanisms of chemotherapeutic resistance deployed by cancers.

RAD51D splice variants and cancer-associated mutations reveal XRCC2 interaction to be critical for homologous recombination.

The proficiency of cancer cells to repair DNA double-strand breaks (DSBs) by homologous recombination (HR) is a key determinant in predicting response to targeted therapies such as PARP inhibitors. The RAD51 paralogs work as multimeric complexes and act downstream of BRCA1 to facilitate HR. Numerous epidemiological studies have linked RAD51 paralog mutations with hereditary cancer predisposition. Despite their substantial links to cancer, RAD51 paralog HR function has remained elusive. Here we identify isoform 1 as the functional isoform of RAD51D, whereas isoform 4 which has a large N-terminal deletion (including the Walker A motif), and isoform 6 which includes an alternate exon in the N-terminus, are non-functional. To determine the importance of this N-terminal region, we investigated the impact of cancer-associated mutations and SNPs in this variable RAD51D N-terminal region using yeast-2-hybrid and yeast-3-hybrid assays to screen for altered protein-protein interactions. We identified two cancer-associated mutations close to or within the Walker A motif (G96C and G107 V, respectively) that independently disrupt RAD51D interaction with XRCC2. We validated our yeast interaction data in human U2OS cells by co-immunoprecipitation and determined the impact of these mutations on HR-proficiency using a sister chromatid recombination reporter assay in a RAD51D knock-out cell line. Our investigation reveals that the interaction of RAD51D with XRCC2 is required for DSB repair. By characterizing the impact of cancer-associated mutations on RAD51D interactions, we aim to develop predictive models for therapeutic sensitivity and resistance in patients who harbor similar mutations in RAD51D.

Fit αß T-cell receptor suppresses leukemogenesis of Pten-deficient thymocytes.

Signaling through the αßT cell receptor (TCR) is a crucial determinant of T-cell fate and can induce two opposite outcomes during thymocyte development: cell death or survival and differentiation. To date, the role played by T-cell receptor in the oncogenic transformation of developing T cells remains unclear. Here we show that human primary T-cell acute lymphoblastic leukemias expressing an αßT cell receptor are frequently deficient for phosphatase and tensin homolog protein (PTEN), and fail to respond strongly to T-cell receptor activation. Using Pten-deficient T-cell acute lymphoblastic leukemia mouse models, we confirm that T-cell receptor signaling is involved in leukemogenesis. We show that abrogation of T-cell receptor expression accelerated tumor onset, while enforced expression of a fit transgenic T-cell receptor led to the development of T-cell receptor-negative lymphoma and delayed tumorigenesis. We further demonstrate that pre-tumoral Pten-deficient thymocytes harboring fit T-cell receptors undergo early clonal deletion, thus preventing their malignant transformation, while cells with unfit T-cell receptors that should normally be deleted during positive selection, pass selection and develop T-cell acute lymphoblastic leukemias. Altogether, our data show that fit T-cell receptor signaling suppresses tumor development mediated by Pten loss-of-function and point towards a role of Pten in positive selection.

RUNX1-dependent RAG1 deposition instigates human TCR-δ locus rearrangement.

V(D)J recombination of TCR loci is regulated by chromatin accessibility to RAG1/2 proteins, rendering RAG1/2 targeting a potentially important regulator of lymphoid differentiation. We show that within the human TCR-α/δ locus, Dδ2-Dδ3 rearrangements occur at a very immature thymic, CD34(+)/CD1a(-)/CD7(+dim) stage, before Dδ2(Dδ3)-Jδ1 rearrangements. These strictly ordered rearrangements are regulated by mechanisms acting beyond chromatin accessibility. Importantly, direct Dδ2-Jδ1 rearrangements are prohibited by a B12/23 restriction and ordered human TCR-δ gene assembly requires RUNX1 protein, which binds to the Dδ2-23RSS, interacts with RAG1, and enhances RAG1 deposition at this site. This RUNX1-mediated V(D)J recombinase targeting imposes the use of two Dδ gene segments in human TCR-δ chains. Absence of this RUNX1 binding site in the homologous mouse Dδ1-23RSS provides a molecular explanation for the lack of ordered TCR-δ gene assembly in mice and may underlie differences in early lymphoid differentiation between these species.

Therapeutic targeting of c-Myc in T-cell acute lymphoblastic leukemia, T-ALL.

T-ALL patients treated with intensive chemotherapy achieve high rates of remission. However, frequent long-term toxicities and relapses into chemotherapy-refractory tumors constitute major clinical challenges which could be met by targeted therapies. c-MYC is a central oncogene in T-ALL, prompting the exploration of the efficacy of MYC inhibitors such as JQ1 (BET-bromodomain inhibitor), and SAHA (HDAC inhibitor). Using a standardized ex vivo drug screening assay, we show here that JQ1 and SAHA show competitive efficiency compared to inhibitors of proteasome, PI3K/AKT/mTOR and NOTCH pathways, and synergize in combination with Vincristine. We also compared for the first time the in vivo relevance of such associations in mice xenografted with human primary T-ALLs. Our data indicate that although treatments combining JQ1 or SAHA with chemotherapeutic regimens might represent promising developments in T-ALL, combinations will need to be tailored to specific subgroups of responsive patients, the profiles of which still remain to be precisely defined.

MYC fails to efficiently shape malignant transformation in T-cell acute lymphoblastic leukemia.

MYC is a potent oncogene involved in ∼70% of human cancers, inducing tumorigenesis with high penetrance and short latency in experimental transgenic models. Accordingly, MYC is recognized as a major driver of T-cell acute lymphoblastic leukemia (T-ALL) in human and zebrafish/mouse models, and uncovering the context by which MYC-mediated malignant transformation initiates and develops remains a considerable challenge. Because MYC is a very complex oncogene, highly dependent on the microenvironment and cell-intrinsic context, we generated transgenic mice (tgMyc(spo)) in which ectopic Myc activation occurs sporadically (<10(-6) thymocytes) within otherwise normal thymic environment, thereby mimicking the unicellular context in which oncogenic alterations initiate human tumors. We show that while Myc(+) clones in tgMyc(spo) mice develop and initially proliferate in thymus and the periphery, no tumor or clonal expansion progress in aging mice (n = 130), suggesting an unexpectedly low ability of Myc to initiate efficient tumorigenesis. Furthermore, to determine the relevance of this observation in human pathogenesis we analyzed a human T-ALL case at diagnosis and relapse using the molecular stigmata of V(D)J recombination as markers of malignant progression; we similarly demonstrate that despite the occurrence of TAL1 and MYC translocations in early thymocyte ontogeny, subsequent oncogenic alterations were required to drive oncogenesis. Altogether, our data suggest that although central to T-ALL, MYC overexpression per se is inefficient in triggering the cascade of events leading to malignant transformation.

Increase in dNTP pool size during the DNA damage response plays a key role in spontaneous and induced-mutagenesis in Escherichia coli.

Exposure of Escherichia coli to UV light increases expression of NrdAB, the major ribonucleotide reductase leading to a moderate increase in dNTP levels. The role of elevated dNTP levels during translesion synthesis (TLS) across specific replication-blocking lesions was investigated. Here we show that although the specialized DNA polymerase PolV is necessary for replication across UV-lesions, such as cyclobutane pyrimidine dimers or pyrimidine(6-4)pyrimidone photoproduct, Pol V per se is not sufficient. Indeed, efficient TLS additionally requires elevated dNTP levels. Similarly, for the bypass of an N-2-acetylaminofluorene-guanine adduct that requires Pol II instead of PolV, efficient TLS is only observed under conditions of high dNTP levels. We suggest that increased dNTP levels transiently modify the activity balance of Pol III (i.e., increasing the polymerase and reducing the proofreading functions). Indeed, we show that the stimulation of TLS by elevated dNTP levels can be mimicked by genetic inactivation of the proofreading function (mutD5 allele). We also show that spontaneous mutagenesis increases proportionally to dNTP pool levels, thus defining a unique spontaneous mutator phenotype. The so-called "dNTP mutator" phenotype does not depend upon any of the specialized DNA polymerases, and is thus likely to reflect an increase in Pol III's own replication errors because of the modified activity balance of Pol III. As up-regulation of the dNTP pool size represents a common physiological response to DNA damage, the present model is likely to represent a general and unique paradigm for TLS pathways in many organisms.

Repurposing lipoic acid changes electron flow in two important metabolic pathways of Escherichia coli.

In bacteria, cysteines of cytoplasmic proteins, including the essential enzyme ribonucleotide reductase (RNR), are maintained in the reduced state by the thioredoxin and glutathione/glutaredoxin pathways. An Escherichia coli mutant lacking both glutathione reductase and thioredoxin reductase cannot grow because RNR is disulfide bonded and nonfunctional. Here we report that suppressor mutations in the lpdA gene, which encodes the oxidative enzyme lipoamide dehydrogenase required for tricarboxylic acid (TCA) cycle functioning, restore growth to this redox-defective mutant. The suppressor mutations reduce LpdA activity, causing the accumulation of dihydrolipoamide, the reduced protein-bound form of lipoic acid. Dihydrolipoamide can then provide electrons for the reactivation of RNR through reduction of glutaredoxins. Dihydrolipoamide is oxidized in the process, restoring function to the TCA cycle. Thus, two electron transfer pathways are rewired to meet both oxidative and reductive needs of the cell: dihydrolipoamide functionally replaces glutathione, and the glutaredoxins replace LpdA. Both lipoic acid and glutaredoxins act in the reverse manner from their normal cellular functions. Bioinformatic analysis suggests that such activities may also function in other bacteria.

Functional plasticity of a peroxidase allows evolution of diverse disulfide-reducing pathways.

In Escherichia coli, the glutathione/glutaredoxin and thioredoxin pathways are essential for the reduction of cytoplasmic protein disulfide bonds, including those formed in the essential enzyme ribonucleotide reductase during its action on substrates. Double mutants lacking thioredoxin reductase (trxB) and glutathione reductase (gor) or glutathione biosynthesis (gshA) cannot grow. Growth of Deltagor DeltatrxB strains is restored by a mutant (ahpC*) of the peroxiredoxin AhpC, converting it to a disulfide reductase that generates reduced glutathione. Here, we show that ahpC* also restores growth to a DeltagshB DeltatrxB strain, which lacks glutathione and accumulates only its precursor gamma-glutamylcysteine (gamma-GC). It suppresses this strain by allowing accumulation of reduced gamma-GC, which can substitute for glutathione. Surprisingly, new ahpC suppressor mutations arose in a DeltagshA DeltatrxB strain lacking both glutathione and gamma-GC, a strain that ahpC* does not suppress. Some of these mutant AhpC proteins channel electrons into the disulfide-reducing pathways via either the thioredoxins or the glutaredoxins without, evidently, the intermediary of glutathione. Our results provide insights into the physiological functioning of the glutathione pathway and reveal surprising plasticity of a peroxidase because different mutant versions of AhpC can channel electrons into the disulfide-reducing pathways by at least four distinct routes. Despite the reductase activity of mutant AhpCs, these various suppressor strains exhibit an oxidizing cytoplasm and accumulate correctly folded disulfide-bonded proteins in their cytoplasm. Proteins most effectively oxidized vary between strains, potentially providing useful tools for expressing different disulfide-bonded proteins.

In vivo requirement for glutaredoxins and thioredoxins in the reduction of the ribonucleotide reductases of Escherichia coli.

Escherichia coli expresses three types of ribonucleotide reductases (RNRs) that utilize the redox chemistry of cysteine to catalyze the reduction of ribonucleotides. Upon reduction, the cysteines form a disulfide bond and must be reduced. The authors present in vivo studies that shed light on the mechanism by which these enzymes are regenerated. The class Ia enzyme, NrdAB, can be reduced by either the thioredoxins 1 and 2 or by glutaredoxin 1. The class Ib enzyme, NrdEF, is reduced in vivo by a dedicated glutaredoxin-like protein, NrdH. Despite its similarities to glutaredoxins, this protein is itself reduced by thioredoxin reductase in vivo. However, in the absence of thioredoxin reductase and NrdH, glutaredoxin 1 can partially replace NrdH. Despite their similar structures, the NrdEF and NrdAB RNRs differ in their abilities to function under low oxygen conditions. With only traces of oxygen present, NrdAB can allow some growth in the absence of the anaerobic enzyme NrdDG. NrdEF cannot. Furthermore, in anaerobiosis, E. coli is dependent for growth on class III RNR, NrdDG, and on having at least one of the two reductive systems, thioredoxin reductase or glutathione reductase. These findings indicate a role for these enzymes either for NrdDG reactivation or some other essential anaerobic process.

Ribonucleotide reductases: influence of environment on synthesis and activity.

Ribonucleotide reductases (RNRs) are enzymes that provide deoxyribonucleotides (dNTPs), the building blocks required for de novo DNA synthesis and repair. They are found in all organisms from prokaryotes to eukaryotes. Interestingly, in the microbial world, several organisms possess the genes encoding two, or even three different RNRs that present different structures and allosteric regulation. The finding of an increasing number of bacterial species that possess more than one RNR might suggest particular functions for these enzymes in different growth conditions. Recent support for this proposal comes from studies indicating that expression and activity of the different RNRs depends on the environment. The oxygen content as well as the redox and oxidative stresses regulate RNR activity and synthesis in various organisms. This regulation has a direct consequence on dNTP pools. An excess of dNTP pools that leads to misincorporation of dNTPs results in genetic abnormalities in eukaryotes as in prokaryotes. In contrast, increased dNTP concentrations help cells to survive under conditions where DNA has been damaged. Hence the use of different RNRs in response to various environmental conditions allows the cell to regulate the amount precisely of dNTP in both a positive and negative manner so that enough, yet not excessive, dNTPs are synthesized.

A novel regulatory mechanism couples deoxyribonucleotide synthesis and DNA replication in Escherichia coli.

We present evidence for a complex regulatory interplay between the initiation of DNA replication and deoxyribonucleotide synthesis. In Escherichia coli, the ATP-bound DnaA protein initiates chromosomal replication. Upon loading of the beta-clamp subunit (DnaN) of the replicase, DnaA is inactivated as its intrinsic ATPase activity is stimulated by the protein Hda. The beta-subunit acts as a matchmaker between Hda and DnaA. Chain elongation of DNA requires a sufficient supply of deoxyribonucleotides (dNTPs), which are produced by ribonucleotide reductase (RNR). We present evidence suggesting that the molecular switch from ATP-DnaA to ADP-DnaA is a critical step coordinating DNA replication with increased deoxyribonucleotide synthesis. Characterization of dnaA and dnaN mutations that result in a constitutively high expression of RNR reveal this mechanism. We propose that the nucleotide bound state of DnaA regulates the transcription of the genes encoding ribonucleotide reductase (nrdAB). Accordingly, the conversion of ATP-DnaA to ADP-DnaA after initiation and loading of the beta-subunit DnaN would allow increased nrdAB expression, and consequently, coordinated RNR synthesis and DNA replication during the cell cycle.

The bacterial ATPase SecA functions as a monomer in protein translocation.

The ATPase SecA drives the post-translational translocation of proteins through the SecY channel in the bacterial inner membrane. SecA is a dimer that can dissociate into monomers under certain conditions. To address the functional importance of the monomeric state, we generated an Escherichia coli SecA mutant that is almost completely monomeric (>99%), consistent with predictions from the crystal structure of Bacillus subtilis SecA. In vitro, the monomeric derivative retained significant activity in various assays, and in vivo, it sustained 85% of the growth rate of wild type cells and reduced the accumulation of precursor proteins in the cytoplasm. Disulfide cross-linking in intact cells showed that mutant SecA is monomeric and that even its parental dimeric form is dissociated. Our results suggest that SecA functions as a monomer during protein translocation in vivo.

Genes regulated by TorR, the trimethylamine oxide response regulator of Shewanella oneidensis.

The torECAD operon encoding the trimethylamine oxide (TMAO) respiratory system of Shewanella oneidensis is positively controlled by the TorS/TorR two-component system when TMAO is available. Activation of the tor operon occurs upon binding of the phosphorylated response regulator TorR to a single operator site containing the direct repeat nucleotide sequence TTCATAN4TTCATA. Here we show that the replacement of any nucleotide of one TTCATA hexamer prevented TorR binding in vitro, meaning that TorR specifically interacts with this DNA target. Identical direct repeat sequences were found in the promoter regions of torR and of the new gene torF (SO4694), and they allowed TorR binding to both promoters. Real-time PCR experiments revealed that torR is negatively autoregulated, whereas torF is strongly induced by TorR in response to TMAO. Transcription start site location and footprinting analysis indicate that the operator site at torR overlaps the promoter -10 box, whereas the operator site at torF is centered at -74 bp from the start site, in agreement with the opposite role of TorR in the regulation of the two genes. Since torF and torECAD are positively coregulated by TorR, we propose that the TorF protein plays a role related to TMAO respiration.

Interactions of glutaredoxins, ribonucleotide reductase, and components of the DNA replication system of Escherichia coli.

A strain of Escherichia coli missing three members of the thioredoxin superfamily, thioredoxins 1 and 2 and glutaredoxin 1, is unable to grow, a phenotype presumed to be due to the inability of cells to reduce the essential enzyme ribonucleotide reductase. Two classes of mutations can restore growth to such a strain. First, we have isolated a collection of mutations in the gene for the protein glutaredoxin 3 that suppress the growth defect. Remarkably, all eight independent mutations alter the same amino acid, methionine-43, changing it to valine, isoleucine, or leucine. From the position of the amino acid changes and their effects, we propose that these alterations change the protein so that its properties are closer to those of glutaredoxin 1. The second means of suppressing the growth defects of the multiply mutant strain was by mutations in the DNA replication genes, dnaA and dnaN. These mutations substantially increase the expression of ribonucleotide reductase, most likely by altering the interaction of the regulatory protein DnaA with the ribonucleotide reductase promoter. Our results suggest that this increase in the concentration of ribonucleotide reductase in the cell allows more effective interaction with glutaredoxin 3, thus restoring an effective pool of deoxyribonucleotides. Our studies present direct evidence that ribonucleotide reductase is the only essential enzyme that requires the three reductive proteins missing in our strains. Our results also suggest an unexpected regulatory interaction between the DnaA and DnaN proteins.

Reconstitution of the trimethylamine oxide reductase regulatory elements of Shewanella oneidensis in Escherichia coli.

Several bacteria can grow by using small organic compounds such as trimethylamine oxide (TMAO) as electron acceptors. In Shewanella species, the TMAO reductase respiratory system is encoded by the torECAD operon. We showed that production of the TMAO reductase of S. oneidensis was induced by TMAO and repressed by oxygen, and we noticed that a three-gene cluster (torSTR) encoding a complex two-component regulatory system was present downstream of the torECAD operon. We introduced the torSTR gene cluster into Escherichia coli and showed that this regulatory gene cluster is involved in TMAO induction of the torE promoter but plays no role in the oxygen control. The TorR response regulator was purified, and gel shift and footprinting experiments revealed that TorR binds to a single region located about 70 bases upstream of the transcription start site of the tor structural operon. By deletion analysis, we confirmed that the TorR operator site is required for induction of the tor structural promoter. As the TMAO regulatory system of S. oneidensis is homologous to that of E. coli, we investigated a possible complementation between the TMAO regulatory components of the two bacteria. Interestingly, TorS(ec), the TMAO sensor of E. coli, was able to transphosphorylate TorR(so), the TMAO response regulator of S. oneidensis.