Recombination Step Research Articles

Statistical analysis of repertoire data demonstrates the influence of microhomology in V(D)J recombination.

Humans rely on diverse adaptive immune receptor repertoires to effectively defend against infections. The receptor generation process, known as V(D)J recombination, is designed to create this diversity by stochastically joining V(D)J gene segments and modifying their junctions through nucleotide deletions and insertions. Previous studies, conducted in vitro, have suggested that short stretches of homologous nucleotides between gene segments can bias these recombination steps. In this study, we explore the extent to which these homologous nucleotides influence V(D)J recombination in humans using statistical inference on large-scale receptor repertoire sequencing data. Our findings reveal that microhomology significantly biases several recombination steps, with important practical implications for the analysis, processing, and interpretation of receptor sequences.

Proximity labeling reveals new functional relationships between meiotic recombination proteins in S. cerevisiae.

Several protein ensembles facilitate crossover recombination and the associated assembly of synaptonemal complex (SC) during meiosis. In yeast, meiosis-specific factors including the DNA helicase Mer3, the "ZZS" complex consisting of Zip4, Zip2, and Spo16, the RING-domain protein Zip3, and the MutSγ heterodimer collaborate with crossover-promoting activity of the SC component, Zip1, to generate crossover-designated recombination intermediates. These ensembles also promote SC formation - the organized assembly of Zip1 with other structural proteins between aligned chromosome axes. We used proximity labeling to investigate spatial relationships between meiotic recombination and SC proteins in S. cerevisiae. We find that recombination initiation and SC factors are dispensable for proximity labeling of Zip3 by ZZS components, but proteins associated with early steps in recombination are required for Zip3 proximity labeling by MutSγ, suggesting that MutSγ joins Zip3 only after a recombination intermediate has been generated. We also find that zip1 separation-of-function mutants that are crossover deficient but still assemble SC fail to generate protein ensembles where Zip3 can engage ZZS and/or MutSγ. The SC structural protein Ecm11 is proximity labeled by ZZS proteins in a Zip4-dependent and Zip1-independent manner, but labeling of Ecm11 by Zip3 and MutSγ requires, at least in part, Zip1. Finally, mass spectrometry analysis of biotinylated proteins in eleven proximity labeling strains uncovered shared proximity targets of SC and crossover-associated proteins, some of which have not previously been implicated in meiotic recombination or SC formation, highlighting the potential of proximity labeling as a discovery tool.

Variable Splitting and Fusing for Image Phase Retrieval.

Phase Retrieval is defined as the recovery of a signal when only the intensity of its Fourier Transform is known. It is a non-linear and non-convex optimization problem with a multitude of applications including X-ray crystallography, microscopy and blind deconvolution. In this study, we address the problem of Phase Retrieval from the perspective of variable splitting and alternating minimization for real signals and seek to develop algorithms with improved convergence properties. An exploration of the underlying geometric relations led to the conceptualization of an algorithmic step aiming to refine the estimate at each iteration via recombination of the separated variables. Following this, a theoretical analysis to study the convergence properties of the proposed method and justify the inclusion of the recombination step was developed. Our experiments showed that the proposed method converges substantially faster compared to other state-of-the-art analytical methods while demonstrating equivalent or superior performance in terms of quality of reconstruction and ability to converge under various setups.

RPS19 and RPL5, the most commonly mutated genes in Diamond Blackfan anemia, impact DNA double-strand break repair.

Diamond Blackfan anemia (DBA) is caused by germline heterozygous loss-of-function pathogenic variants (PVs) in ribosomal protein (RP) genes, most commonly RPS19 and RPL5 . In addition to red cell aplasia, individuals with DBA are at increased risk of various cancers. Importantly, the mechanism(s) underlying cancer predisposition are poorly understood. We found that DBA patient-derived lymphoblastoid cells had persistent γ-H2AX foci following ionizing radiation (IR) treatment, suggesting DNA double-strand break (DSB) repair defects. RPS19- and RPL5-knocked down (KD) CD34+ cells had delayed repair of IR-induced DSBs, further implicating these RPs in DSB repair. Assessing the impact of RPS19- and RPL5-KD on specific DSB repair pathways, we found RPS19-KD decreased the efficiency of pathways requiring extensive end-resection, whereas RPL5-KD increased end-joining pathways. Additionally, RAD51 was reduced in RPS19- and RPL5-KD and RPS19- and RPL5-mutated DBA cells, whereas RPS19-deficient cells also had a reduction in PARP1 and BRCA2 proteins. RPS19-KD cells had an increase in nuclear RPA2 and a decrease in nuclear RAD51 foci post-IR, reflective of alterations in early, critical steps of homologous recombination. Notably, RPS19 and RPL5 interacted with poly(ADP)-ribose chains noncovalently, were recruited to DSBs in a poly(ADP)-ribose polymerase activity-dependent manner, and interacted with Ku70 and histone H2A. RPL5's recruitment, but not RPS19's, also required p53, suggesting that RPS19 and RPL5 directly participate in DSB repair via different pathways. We propose that defective DSB repair arising from haploinsufficiency of these RPs may underline the cancer predisposition in DBA.

RPS15 Coordinates with CtIP to Facilitate Homologous Recombination and Confer Therapeutic Resistance in Breast Cancer.

The repair of DNA double-strand breaks (DSBs) through homologous recombination (HR) is vital for maintaining the stability and integrity of the genome. RNA binding proteins (RBPs) intricately regulate the DNA damage repair process, yet the precise molecular mechanisms underlying their function remain incompletely understood. In this study, we highlight the pivotal role of RPS15, a representative RBP, in homologous recombination repair. Specifically, we demonstrate that RPS15 promotes DNA end resection, a crucial step in homologous recombination. Notably, we identify an interaction between RPS15 and CtIP, a key factor in homologous recombination repair. This interaction is essential for CtIP recruitment to DSB sites, subsequent RPA coating, and RAD51 replacement, all critical steps in efficient homologous recombination repair and conferring resistance to genotoxic treatments. Functionally, suppressing RPS15 expression sensitizes cancer cells to X-ray radiation and enhances the therapeutic synergistic effect of PARP1 inhibitors in breast cancer cells. In summary, our findings reveal that RPS15 promotes DNA end resection to ensure effective homologous recombination repair, suggesting its potential as a therapeutic target in cancer treatment.

Early selection efficiency in a progeny test of timber species Cordia trichotoma (Vell.) Arráb. ex Steud

One of the key obstacles in forest breeding routines is the time-consuming steps of selection and recombination of superior genotypes owing to the long life cycle of trees. Considering the increase in global temperature and constant climate alterations, the quick selection of genetic materials is crucial to ensure the effectiveness of genetic improvement programs. Early selection studies on other forest species are scarce, barring eucalyptus and pine, which are well-studied crops. In this context, this study aimed to understand the efficiency of early selection in a progeny test of Cordia trichotoma (Vell.) Arráb. ex Steud to evaluate age-age trends, genetic variability, and potential gains. A progeny test was conducted and measured at three, six, and eight years of age for growth traits, diameter, and volume increment. The experiment included 21 open-pollinated families of C. trichotoma arranged in a 4 × 4 m experimental design grid with a single-tree plot distributed into 10 blocks. The genetic correlations among the three measurements for genitor selection were greater than 0.90 for all the traits. For every ten progenies selected at three years of age, approximately seven were determined to be similarly top-ranked at eight years of age, taking into account an early selection assertiveness rate of approximately 0.70 for progenies. Considering the high age-age genetic correlation found in the C. trichotoma progeny test, there is a significant possibility of improving genetic gains per unit of time through early selection. The general mean of volume increment was 18 m3 ha−1 year−1, with productive genotypes showing a volume greater than 30 m3 ha−1 year−1 and representing an estimated general gain of 66 %. Superior genotypes of C. trichotoma are highly competitive in terms of volumetric yield, can generate considerable gains, and are used in the forest industry. Early selection of C. trichotoma is efficient, with a high rate of assertiveness.

(Keynote) Light Driven H2 Generation in Pt-Tipped CdS Nanorods: Dependence on the Pt Size and CdS Rod Length

Colloidal quantum confined semiconductor-metal nano-heterostructures are a promising class of photocatalysts for solar energy conversion. In these photocatalysts, the semiconductor domain serves as the light absorber and the metal as the catalyst. Such photocatalysts combine the superior light absorption and charge transport properties of the semiconductor with the superior catalytic activity and selectivity of the metal. Furthermore, both domains can be independently tuned to enhance the photocatalytic performance of the heterostructure. Among various semiconductor/metal heterostructures, metal-tipped colloidal semiconductor nanorods have attracted extensive interest because they have been reported to have high quantum efficiencies of light driven H2 generation and their morphology can be systematically tuned. The overall light driven H2 generation process involves multiple elementary charge separation and recombination steps in the semiconductor and across the semiconductor/metal interface as well as proton-coupled electron transfer reactions at the catalytic center. The change of the semiconductor or metal domains can often have effects on multiple competing processes involved in the overall reaction. As a result of these complexities, the mechanisms for the morphological dependence of the observed H2 generation efficiencies are not fully understood, hindering the rational design of these photocatalysts. In this talk, we use Pt tipped CdS nanorods (CdS-Pt) as a model system to examine the effect of Pt size and CdS rod length on their light driven H2 generation efficiency. We show that increasing the Pt particle size increases the overall H2 generation quantum efficiency through both increasing the rate of electron transfer from the CdS to Pt and enhancing the efficiency of water/proton reduction; H2 generation efficiency increases at longer CdS rod length by suppression of charge recombination across the Pt/CdS interface. Furthermore, because of rapid trapping of valence band holes, multi-exciton states in CdS nanorods can be long lived, enabling efficient multi-electron transfer to the Pt. Our work demonstrates that through systematic in situ study of elementary processes involved in the overall H2 generation, it is possible to rationally design and improve semiconductor-metal hybrid photocatalysts.

DSB-induced oxidative stress: Uncovering crosstalk between DNA damage response and cellular metabolism

While that ROS causes DNA damage is well documented, there has been limited investigation into whether DNA damages and their repair processes can conversely induce oxidative stress. By generating a site-specific DNA double strand break (DSB) via I-SceI endonuclease expression in S. cerevisiae without damaging other cellular components, this study demonstrated that DNA repair does trigger oxidative stress. Deleting genes participating in the initiation of the resection step of homologous recombination (HR), like the MRX complex, resulted in stimulation of ROS. In contrast, deleting genes acting downstream of HR resection suppressed ROS levels. Additionally, blocking non-homologous end joining (NHEJ) also suppressed ROS. Further analysis identified Rad53 as a key player that relays DNA damage signals to alter redox metabolism in an HR-specific manner. These results suggest both HR and NHEJ can drive metabolism changes and oxidative stress, with NHEJ playing a more prominent role in ROS stimulation. Further analysis revealed a correlation between DSB-induced ROS increase and enhanced activity of NADPH oxidase Yno1 and various antioxidant enzymes. Deleting the antioxidant gene SOD1 induced synthetic lethality in HR-deficient mutants like mre11Δ and rad51Δ upon DSB induction. These findings uncover a significant interplay between DNA repair mechanisms and cellular metabolism, providing insights into understanding the side effects of genotoxic therapies and potentially aiding development of more effective cancer treatment strategies.

An acidic loop in the forkhead-associated domain of the yeast meiosis-specific kinase Mek1 interacts with a specific motif in a subset of Mek1 substrates.

The meiosis-specific kinase Mek1 regulates key steps in meiotic recombination in the budding yeast, Saccharomyces cerevisiae. MEK1 limits resection at double-strand break (DSB) ends and is required for preferential strand invasion into homologs, a process known as interhomolog bias. After strand invasion, MEK1 promotes phosphorylation of the synaptonemal complex protein Zip1 that is necessary for DSB repair mediated by a crossover-specific pathway that enables chromosome synapsis. In addition, Mek1 phosphorylation of the meiosis-specific transcription factor, Ndt80, regulates the meiotic recombination checkpoint that prevents exit from pachytene when DSBs are present. Mek1 interacts with Ndt80 through a 5-amino acid sequence, RPSKR, located between the DNA-binding and activation domains of Ndt80. AlphaFold Multimer modeling of a fragment of Ndt80 containing the RPSKR motif and full-length Mek1 indicated that RPSKR binds to an acidic loop located in the Mek1 FHA domain, a noncanonical interaction with this motif. A second protein, the 5'-3' helicase Rrm3, similarly interacts with Mek1 through an RPAKR motif and is an in vitro substrate of Mek1. Genetic analysis using various mutants in the MEK1 acidic loop validated the AlphaFold model, in that they specifically disrupt 2-hybrid interactions with Ndt80 and Rrm3. Phenotypic analyses further showed that the acidic loop mutants are defective in the meiotic recombination checkpoint and, in certain circumstances, exhibit more severe phenotypes compared to the NDT80 mutant with the RPSKR sequence deleted, suggesting that additional, as yet unknown, substrates of Mek1 also bind to Mek1 using an RPXKR motif.

Multi-step control of homologous recombination via Mec1/ATR suppresses chromosomal rearrangements

The Mec1/ATR kinase is crucial for genome stability, yet the mechanism by which it prevents gross chromosomal rearrangements (GCRs) remains unknown. Here we find that in cells with deficient Mec1 signaling, GCRs accumulate due to the deregulation of multiple steps in homologous recombination (HR). Mec1 primarily suppresses GCRs through its role in activating the canonical checkpoint kinase Rad53, which ensures the proper control of DNA end resection. Upon loss of Rad53 signaling and resection control, Mec1 becomes hyperactivated and triggers a salvage pathway in which the Sgs1 helicase is recruited to sites of DNA lesions via the 911-Dpb11 scaffolds and phosphorylated by Mec1 to favor heteroduplex rejection and limit HR-driven GCR accumulation. Fusing an ssDNA recognition domain to Sgs1 bypasses the requirement of Mec1 signaling for GCR suppression and nearly eliminates D-loop formation, thus preventing non-allelic recombination events. We propose that Mec1 regulates multiple steps of HR to prevent GCRs while ensuring balanced HR usage when needed for promoting tolerance to replication stress.

An acidic loop in the FHA domain of the yeast meiosis-specific kinase Mek1 interacts with a specific motif in a subset of Mek1 substrates.

The meiosis-specific kinase Mek1 regulates key steps in meiotic recombination in the budding yeast, Saccharomyces cerevisiae. MEK1 limits resection at the double strand break (DSB) ends and is required for preferential strand invasion into homologs, a process known as interhomolog bias. After strand invasion, MEK1 promotes phosphorylation of the synaptonemal complex protein Zip1 that is necessary for DSB repair mediated by a crossover specific pathway that enables chromosome synapsis. In addition, Mek1 phosphorylation of the meiosis-specific transcription factor, Ndt80, regulates the meiotic recombination checkpoint that prevents exit from pachytene when DSBs are present. Mek1 interacts with Ndt80 through a five amino acid sequence, RPSKR, located between the DNA binding and activation domains of Ndt80. AlphaFold Multimer modeling of a fragment of Ndt80 containing the RPSKR motif and full length Mek1 indicated that RPSKR binds to an acidic loop located in the Mek1 FHA domain, a non-canonical interaction with this motif. A second protein, the 5'-3' helicase Rrm3, similarly interacts with Mek1 through an RPAKR motif and is an in vitro substrate of Mek1. Genetic analysis using various mutants in the MEK1 acidic loop validated the AlphaFold model, in that they specifically disrupt two-hybrid interactions with Ndt80 and Rrm3. Phenotypic analyses further showed that the acidic loop mutants are defective in the meiotic recombination checkpoint, and in certain circumstances exhibit more severe phenotypes compared to the NDT80 mutant with the RPSKR sequence deleted, suggesting that additional, as yet unknown, substrates of Mek1 also bind to Mek1 using an RPXKR motif.

The asymptotic solution of near-limit chain-branching premixed flames with the Zel’dovich–Liñán two-step mechanism in the linear and fast recombination regime

Near-limit characteristics of chain-branching premixed flames is investigated numerically and asymptotically by employing the Zel’dovich–Liñán two-step mechanism with linear and (sufficiently) fast recombination, for which both chain-branching and -recombination steps take place in a thin reactive layer of O(β−1), with β being the chain-branching Zel’dovich number. The numerical and asymptotic results reveal that the rate-ratio eigenvalue r between the chain branching and recombination steps approaches a minimum critical value of rmin→e as the chain-recombination step becomes infinitely fast. The existence of the finite asymptotic value of r=e indicates that no steadily propagating flame solution can be found for fuel concentrations lower than its critical value, corresponding to the criticality of r=e, thereby explaining the (lean) flammability limit. This intrinsic flammability characteristics with linear recombination is the most outstanding contrast to the quadratic recombination counterpart, for which flammability is achieved only with external heat loss added to the model.Novelty and significance statementNear-limit combustion characteristics of chain-branching flames is investigated numerically and asymptotically by employing the Zel’dovich–Linan two-step chemical kinetics with linear and fast recombination. This work demonstrates that the rate-ratio eigenvalue between the chain-branching and -recombination steps asymptotically approaches a critical minimum value, corresponding to the flammability limit. The existence of flammability limit with the linear recombination is found to be the most significant contrast to the quadratic recombination model, which is incapable of predicting flammability limit. By this study, a formal asymptotic procedure, by which the crossover temperature and lean flammability limit condition can be determined without assuming a steady-state approximation of intermediate species, is established and can be applied to analyze flame structures with multi-step reduced mechanisms.

Unraveling Enyne Bonding via Dehydrogenation–Hydrogenation Processes in On‐Surface Synthesis with Terminal Alkynes

AbstractOn‐surface reactions of terminal alkynes in ultrahigh vacuum have attracted widespread attention due to their high technological promise. However, employing different precursors and substrate materials often intricate reaction schemes appear far from being well‐understood. Thus, recent investigations of alkyne coupling on noble metal surfaces suggest non‐dehydrogenative scenarios, contradicting earlier reports. Herein, the study employs noncontact atomic force microscopy (nc‐AFM) with high spatial resolution to conclusively characterize exemplary alkyne coupling products. Contrary to initial interpretations proposing dehydrogenative homocoupling on Ag(111), bond‐resolved AFM imaging reveals the expression of enyne motifs. Based on complementary, extensive density functional theory calculations, the pertaining reaction mechanisms are explored. It is proposed that enyne formation initiates with a direct carbon–carbon coupling between two alkyne groups, followed by surface‐assisted dehydrogenation‐hydrogenation processes. Thereby consecutive steps of atomic hydrogen cleavage, surface migration and recombination to a different carbon atom enable bridging via carbon–carbon double bonding. The new results shed light on subtle, but crucial surface‐mediated hydrogen transfer processes involved in the chemical bond formation, which are suggested to be of general relevance in on‐surface synthesis.

The Asymptotic Structure of Strained Chain-Branching Premixed Flames Under Nonadiabatic Conditions

ABSTRACT The present paper, which investigates the chain-branching premixed-flame dynamics under nonadiabatic conditions, is the third and final contribution to a series of studies on premixed flames with the modified Zel’dovich-Liñán two-step mechanism under the three most prevailing physical influences, namely (i) flame stretch, (ii) differential diffusion, and (iii) heat loss to or gain from the flame downstream. Asymptotic analysis for the chain-branching premixed-flame structure is carried out within the framework of the intermediate recombination regime, in which the chain-recombination layer with a characteristic thickness of O ( β − 1 / 2 ) is asymptotically thicker than the inner chain-branching layer but thinner than the outer convective-diffusive layer. The combustion characteristics are presented by the M – Δ plots, where the specific reaction intensity M is a measure of the reaction intensity and the reduced recombination Damköhler number Δ is a measure of the inverse of the flame stretch. The M – Δ plots are found to exhibit a striking resemblance to the AEA counterparts of Libby and Williams (1983), in that the S-shaped M – Δ curves emerge for sufficiently large downstream heat losses. The Damköhler number ratio D I / D II is found to be another key parameter controlling the chain-branching flame dynamics. The greater D I / D II , the slower the recombination reaction and the thicker the recombination layer tends to be, which results in a reduced overall nonlinearity for the Zel’dovich-Liñán two-step mechanism. Consequently, abrupt extinction is less likely observable in the much slower recombination regime. Combining with the results of the previous two papers by the authors, the overall combustion characteristics of strained chain-branching premixed flames with the Zel’dovich-Liñán two-step kinetics is found to be in qualitatively good agreement with that of one-step AEA, unless the recombination step is too slow or too fast to be reasonably described by the intermediate recombination regime.

Human Rad51 Protein Requires Higher Concentrations of Calcium Ions for D-Loop Formation than for Oligonucleotide Strand Exchange.

Human Rad51 protein (HsRad51)-promoted DNA strand exchange, a crucial step in homologous recombination, is regulated by proteins and calcium ions. Both the activator protein Swi5/Sfr1 and Ca2+ ions stimulate different reaction steps and induce perpendicular DNA base alignment in the presynaptic complex. To investigate the role of base orientation in the strand exchange reaction, we examined the Ca2+ concentration dependence of strand exchange activities and structural changes in the presynaptic complex. Our results show that optimal D-loop formation (strand exchange with closed circular DNA) required Ca2+ concentrations greater than 5 mM, whereas 1 mM Ca2+ was sufficient for strand exchange between two oligonucleotides. Structural changes indicated by increased fluorescence intensity of poly(dεA) (a poly(dA) analog) reached a plateau at 1 mM Ca2+. Ca2+ > 2 mM was required for saturation of linear dichroism signal intensity at 260 nm, associated with rigid perpendicular DNA base orientation, suggesting a correlation with the stimulation of D-loop formation. Therefore, Ca2+ exerts two different effects. Thermal stability measurements suggest that HsRad51 binds two Ca2+ ions with KD values of 0.2 and 2.5 mM, implying that one step is stimulated by one Ca2+ bond and the other by two Ca2+ bonds. Our results indicate parallels between the Mg2+ activation of RecA and the Ca2+ activation of HsRad51.

Abstract 7237: Peptide-mRNA complex mediated restoration of BRCA-1 tumor suppressor function in BRCA-1 mutated cancers as a new therapeutic strategy

Abstract BRCA1 and BRCA-2 proteins are involved in the repair of DNA damage such as double-strand breaks during S and G2 phases, by intervening in homologous recombination (HR) steps. Breast cancers arising from BRCA-1 mutation carriers (10% of BC) and from epigenetically silenced BRCA-1 (30% of sporadic BC) are associated with a lack of expression and/or function of BRCA-1, which induces genomic instability. Herein, we describe a new potent strategy combining mRNAs with a tumor selective peptide nanocarrier, to selectively rescue BRCA-1 function as potential therapeutic approach in cancers. Proprietary BRCA-1 derived mRNA were complexed with proprietary short amphipathic peptides and the resulting nanoparticles were evaluated on several breast cancer cells, harboring different BRCA-1 alterations. Sensitivity to Veliparib (PARPi) following the nanoparticle treatments were evaluated on PARPI resistant and PARPi sensitive cells. The recruitment of RAD51 to sites of double stranded DNA break was quantified by immunofluorescence. In-vivo efficacy of varying sequences of BRCA-1 mRNA-peptide nanoparticles (2.0 mg/kg, weekly, IV; ADGN-901, -902, -903) and veliparib (50mg/kg, daily, oral) were evaluated on breast cancer (HCC1937-BRCA-1 deficient, PARPi resistant) mouse xenografts. We showed that ADGN-901 inhibits the proliferation of BRCA-1 deficient cell lines from 20 to 60% depending on the type of BRCA-1 alterations. ADGN-902, specifically reduces the proliferation of cells expressing N-terminal truncated BRCA-1, by 50 to 60% and ADGN-903 inhibits proliferation >60% of all cells harboring defects in BRCA-1. Interestingly, ADGN-901 and ADGN-903 were able to resensitize the PARPi resistant cells to veliparib inhibition. We demonstrated that ADGN-903 restores BRCA-1 function in homologous recombination. ADGN-903 prevents RAD51 recruitment to the damage site by reducing RAD51 protein level as well as formation of RAD51 foci following irradiation. IV administration of ADGN-901 and ADGN-903 (2.0 mg/kg) had a strong inhibitory effect on HCC-1937 tumor growth while ADGN-902 and veliparib were inactive. ADGN-901 reduced tumor growth by 95% and ADGN-903 abolished tumor growth with strong tumor regressions. ADGN-901 and ADGN-903, were also evaluated on large tumors of about 500-600 mm3 and were highly effective in preventing further progression and achieving shrinkage of large tumors. ADGN-901 and ADGN-903 treatments are well tolerated, without inducing clinical toxicity. BRCA-1 mRNA-peptide nanoparticles are effective in rescuing BRCA-1 function and inducing tumor regressions in BRCA-1 altered breast cancer cells and tumors. Our study provides a proof-of-concept that restoration of wild type BRCA-1 expression could be a potent therapeutic strategy for BRCA-1 altered tumors whether or not they are PARPi resistant. Citation Format: Gilles Divita, Audrey Grunenberger, Léa Cabrera, Nathalie Durany, Neil Desai. Peptide-mRNA complex mediated restoration of BRCA-1 tumor suppressor function in BRCA-1 mutated cancers as a new therapeutic strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7237.

SMARCAL1 ubiquitylation controls its association with RPA-coated ssDNA and promotes replication fork stability.

Impediments in replication fork progression cause genomic instability, mutagenesis, and severe pathologies. At stalled forks, RPA-coated single-stranded DNA (ssDNA) activates the ATR kinase and directs fork remodeling, 2 key early events of the replication stress response. RFWD3, a recently described Fanconi anemia (FA) ubiquitin ligase, associates with RPA and promotes its ubiquitylation, facilitating late steps of homologous recombination (HR). Intriguingly, RFWD3 also regulates fork progression, restart and stability via poorly understood mechanisms. Here, we used proteomics to identify putative RFWD3 substrates during replication stress in human cells. We show that RFWD3 interacts with and ubiquitylates the SMARCAL1 DNA translocase directly in vitro and following DNA damage in vivo. SMARCAL1 ubiquitylation does not trigger its subsequent proteasomal degradation but instead disengages it from RPA thereby regulating its function at replication forks. Proper regulation of SMARCAL1 by RFWD3 at stalled forks protects them from excessive MUS81-mediated cleavage in response to UV irradiation, thereby limiting DNA replication stress. Collectively, our results identify RFWD3-mediated SMARCAL1 ubiquitylation as a novel mechanism that modulates fork remodeling to avoid genome instability triggered by aberrant fork processing.

Trans-complementation by the RecB nuclease domain of RecBCD enzyme reveals new insight into RecA loading upon χ recognition.

The loading of RecA onto ssDNA by RecBCD is an essential step of RecBCD-mediated homologous recombination. RecBCD facilitates RecA-loading onto ssDNA in a χ-dependent manner via its RecB nuclease domain (RecBn). Before recognition of χ, RecBn is sequestered through interactions with RecBCD. It was proposed that upon χ-recognition, RecBn undocks, allowing RecBn to swing out via a contiguous 70 amino acid linker to reveal the RecA-loading surface, and then recruit and load RecA onto ssDNA. We tested this hypothesis by examining the interactions between RecBn (RecB928-1180) and truncated RecBCD (RecB1-927CD) lacking the nuclease domain. The reconstituted complex of RecB1-927CD and RecBn is functional in vitro and in vivo. Our results indicate that despite being covalently severed from RecB1-927CD, RecBn can still load RecA onto ssDNA, establishing that RecBn does not function while only remaining tethered to the RecBCD complex via the linker. Instead, RecBCD undergoes a χ-induced intramolecular rearrangement to reveal the RecA-loading surface.

Tracking Charge Migration with Frequency-Matched Strobo-Spectroscopy.

We present frequency-matched strobo-spectroscopy (FMSS) of charge migration (CM) in bromobutadiyne, simulated with time-dependent density functional theory. CM + FMSS is a pump-probe scheme that uses a frequency-matched high harmonic generation (HHG)-driving laser as an independent probe step, following the creation of a localized hole on the bromine atom that induces CM dynamics. We show that the delay-dependent harmonic yield tracks the phase of the CM dynamics through its sensitivity to the amount of electron density on the bromine end of the molecule. FMSS takes advantage of the intrinsic attosecond time resolution of the HHG process in which different harmonics are emitted at different times and thus probe different locations of the electron hole. Finally, we show that the CM-induced modulation of the HHG signal is dominated by the recombination step of the HHG process, with a negligible contribution from the ionization step.

Abstract C102: mRNA-mediated restoration of BRCA-1 tumor suppressor function prevents tumor growth and reverses resistance to PARP inhibitors in triple-negative breast cancers

Abstract BRCA1 and BRCA2 proteins are involved in the repair of DNA damage such as double-strand breaks during S and G2 phases, by intervening in homologous recombination (HR) steps. Breast cancers arising from BRCA-1 mutation carriers, up to 10% BC, are associated with a lack of expression and/or function of BRCA-1 protein, which induces genomic instability. Considering the major role of BRCA1 gene in DNA repair process, therapeutic approaches using cytotoxic agents such as the platinum and PARP inhibitors are used as treatments. However, hypersensitivity to PARPi is limited in the clinic by acquired resistance after restoration of HR. We have developed a new potent strategy combining mRNAs with a tumor selective nanocarrier, to selectively rescue BRCA-1 functions and restore PARPi sensitivity, as potential therapeutic approach in cancers. ADGN-technology is based on short amphipathic peptides that form stable neutral nanoparticles with mRNA. ADGN-901, -902 , -903 nanoparticles containing mRNA derived from BRCA-1 were evaluated on breast cancer cells, harboring different BRCA-1 deletion and mutations. Cell proliferation, cell cycle level and apoptosis activation were determined by flow cytometry and Tunel assay. BRCA-1 and RAD51 protein levels were evaluated by western blot. Sensitivity to Veliparib (PARPi) following ADGN-mRNA treatment was evaluated on PARPI resistance and PARPi sensitive cells. The recruitment of RAD51 to sites of double stranded DNA break was quantified by immunofluorescence. In-vivo efficacy of IV-administered ADGN-BRCA NPs (2.0 mg/kg) was evaluated on breast (HCC1937-BRCA-1 deficient, PARPi resistant) cancer mouse xenografts. We have selected specific mRNA sequences encoding for different domains of BRCA-1 protein. We showed than ADGN-901 mediated rescue of BRCA-1 wild type expression inhibits the proliferation of BRCA-1 deficient cell lines from 20 to 60% depending on the type of BRCA-1 alterations. ADGN-902, specifically reduces the proliferation of cells expressing N-terminal truncated BRCA-1, by 50 to-60% and ADGN-903 markedly delay (>60%) the growth of all cells harboring defect in BRCA-1. In contrast, no significant cytotoxicity related to vehicle was observed. ADGN-901 and ADGN-903 resensitize the resistant cells to PARPi Veliparib inhibition. We demonstrated that the expression of domain of BRCA-1 encoded by ADGN-903 mRNA, appears to prevent RAD51 recruitment to the damage site by reducing RAD51 protein level as well as formation of RAD51 foci following irradiation. Intravenous-administration ADGN-901 and ADGN-903 (2.0mg/kg) prevented HCC-1937 tumor growth. ADGN-901 reduced tumor growth by 95% and ADGN-903 resulted in a tumor regression of 50%. ADGN-901 and ADGN-903 treatments are well tolerated, without inducing clinical toxicity. ADGN-BRCA nanoparticles are effective in rescuing BRCA-1 functions in Breast cancer cells. Our study provides a proof-of-concept that restoration of the expression of the wild type form of BRCA-1 could be combined together with PARP inhibition for potent combinatorial cancer treatment. Citation Format: Gilles DIVITA, Audrey Grunenberger, Léa Cabrera, Nathalie Durany, Khadidja Boularache, Veronique Josserand, Neil Desai. mRNA-mediated restoration of BRCA-1 tumor suppressor function prevents tumor growth and reverses resistance to PARP inhibitors in triple-negative breast cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C102.