RAD51 Paralogs Research Articles

A Rare Case of Triple-Negative Breast Cancer with RAD51D Gene Mutation

Background: The heterogeneity of breast cancer (BC) subtypes poses a significant challenge, with carcinogenesis involving multiple stages and genes, including proto-oncogenes, tumor suppressor genes, and DNA repair genes. Next-generation sequencing has expanded access to multigene panels, such as RAD51 paralogs, which increase the risk of ovarian cancer and possibly triple-negative (TN) BC. Case presentation: We present a rare case of a 45-year-old woman with TNBC and a RAD51D gene mutation. Mammography and breast ultrasonography revealed an irregular, 30 mm hypoechoic area and dystrophic calcifications in the right breast. Immunohistochemistry showed a lack of expression of ER, RP, HER-2, and P53, with 50% of neoplastic cell nuclei positive for Ki-67. Next-generation sequencing revealed a mutation in RAD51D and MUYTH genes. The patient underwent partial mastectomy, chemotherapy, and prophylactic mastectomy. Conclusion: Genetic analysis is crucial for identifying specific mutations contributing to TNBC development. Current preventive interventions primarily address BRCA1 and BRCA2 mutations, following established guidelines.

FIGL1 attenuates meiotic interhomolog repair and is counteracted by the RAD51 paralog XRCC2 and the chromosome axis protein ASY1 during meiosis.

Two recombinases, RAD51 and DMC1, catalyze meiotic break repair to ensure crossovers (COs) between homologous chromosomes (interhomolog) rather than between sisters (intersister). FIDGETIN-LIKE-1 (FIGL1) downregulates both recombinases. However, the understanding of how FIGL1 functions in meiotic repair remains limited. Here, we discover new genetic interactions of Arabidopsis thaliana FIGL1 that are important in vivo determinants of meiotic repair outcome. In figl1 mutants, compromising RAD51-dependent repair, either through the loss of RAD51 paralogs (RAD51B or XRCC2) or RAD54 or by inhibiting RAD51 catalytic activity, results in either unrepaired breaks or meiotic CO defects. Further, XRCC2 physically interacts with FIGL1 and partially counteracts FIGL1 activity for RAD51 focus formation. Our data indicate that RAD51-mediated repair mechanisms compensate FIGL1 dysfunction. FIGL1 is not necessary for intersister repair in dmc1 but is essential for the completion of meiotic repair in mutants such as asy1 that have impaired DMC1 functions and interhomolog bias. We show that FIGL1 attenuates interhomolog repair, and ASY1 counteracts FIGL1 to promote interhomolog recombination. Altogether, this study underlines that multiple factors can counteract FIGL1 activity to promote accurate meiotic repair.

RAD51 Paralogs and RAD51 Paralog Complexes BCDX2 and CX3 Interact with BRCA2.

Homologous recombination (HR) is an important mechanism for repairing DNA double-strand breaks (DSBs) and preserving genome integrity. Pathogenic mutations in the HR proteins BRCA2 and the RAD51 paralogs predispose individuals to breast, ovarian, pancreatic, and prostate cancer. The RAD51 paralogs: RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3 form two complexes RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) and RAD51C-XRCC3 (CX3). Similar to BRCA2, loss of RAD51 paralog functions in mammalian cells lead to chromosomal abnormalities, growth defects, disrupted RAD51 foci formation, and PARP inhibitor sensitivity. Despite significant effort over the past three decades, the specific molecular functions of the human RAD51 paralogs have remained elusive due to technical challenges such as low protein expression in human cell lines and instability of the purified proteins. Recent studies have determined the molecular structures of the BCDX2 and CX3 complexes dramatically enhancing our understanding of these challenging proteins. Using multiple approaches, we demonstrate that the RAD51 paralogs interact with BRCA2 at two distinct interaction hubs located in the BRC repeats and the DNA binding domain. We confirm, using a yeast 3-hybrid approach, that human RAD51 paralogs interact directly with BRC repeats one and two (BRC1-2) of BRCA2. Because of the dynamic nature of the RAD51B C-terminal domain (CTD), identified in the recently solved cryo-EM structures, we focused on elucidating the interaction with RAD51B. We determined that BRCA2 interacts with the CTD of RAD51B and not the N-terminal domain (NTD) that is involved in stacking interactions with RAD51C and RAD51D. Furthermore, the interaction with RAD51B is dependent upon an FxxA motif located on a surface exposed region of the CTD. Our study has identified novel interactions between the RAD51 paralogs and BRCA2 and further demonstrated that a previously unrecognized FxxA motif located within a mobile element of RAD51B is critical for the interaction.

Mechanism of BCDX2-mediated RAD51 nucleation on short ssDNA stretches and fork DNA.

Homologous recombination (HR) factors are crucial for DSB repair and processing stalled replication forks. RAD51 paralogs, including RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, have emerged as essential tumour suppressors, forming two subcomplexes, BCDX2 and CX3. Mutations in these genes are associated with cancer susceptibility and Fanconi anaemia, yet their biochemical activities remain unclear. This study reveals a linear arrangement of BCDX2 subunits compared to the RAD51 ring. BCDX2 shows a strong affinity towards single-stranded DNA (ssDNA) via unique binding mechanism compared to RAD51, and a contribution of DX2 subunits in binding branched DNA substrates. We demonstrate that BCDX2 facilitates RAD51 loading on ssDNA by suppressing the cooperative requirement of RAD51 binding to DNA and stabilizing the filament. Notably, BCDX2 also promotes RAD51 loading on short ssDNA and reversed replication fork substrates. Moreover, while mutants defective in ssDNA binding retain the ability to bind branched DNA substrates, they still facilitate RAD51 loading onto reversed replication forks. Our study provides mechanistic insights into how the BCDX2 complex stimulates the formation of BRCA2-independent RAD51 filaments on short stretches of ssDNA present at ssDNA gaps or stalled replication forks, highlighting its role in genome maintenance and DNA repair.

The human Shu complex promotes RAD51 activity by modulating RPA dynamics on ssDNA

Templated DNA repair that occurs during homologous recombination and replication stress relies on RAD51. RAD51 activity is positively regulated by BRCA2 and the RAD51 paralogs. The Shu complex is a RAD51 paralog-containing complex consisting of SWSAP1, SWS1, and SPIDR. We demonstrate that SWSAP1-SWS1 binds RAD51, maintains RAD51 filament stability, and enables strand exchange. Using single-molecule confocal fluorescence microscopy combined with optical tweezers, we show that SWSAP1-SWS1 decorates RAD51 filaments proficient for homologous recombination. We also find SWSAP1-SWS1 enhances RPA diffusion on ssDNA. Importantly, we show human sgSWSAP1 and sgSWS1 knockout cells are sensitive to pharmacological inhibition of PARP and APE1. Lastly, we identify cancer variants in SWSAP1 that alter Shu complex formation. Together, we show that SWSAP1-SWS1 stimulates RAD51-dependent high-fidelity repair and may be an important new cancer therapeutic target.

RAD51 paralogs synergize with RAD51 to protect reversed forks from cellular nucleases.

Fork reversal is a conserved mechanism to prevent stalled replication forks from collapsing. Formation and protection of reversed forks are two crucial steps in ensuring fork integrity and stability. Five RAD51 paralogs, namely, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, which share sequence and structural similarity to the recombinase RAD51, play poorly defined mechanistic roles in these processes. Here, using purified BCDX2 (RAD51BCD-XRCC2) and CX3 (RAD51C-XRCC3) complexes and in vitro reconstituted biochemical systems, we mechanistically dissect their functions in forming and protecting reversed forks. We show that both RAD51 paralog complexes lack fork reversal activities. Whereas CX3 exhibits modest fork protection activity, BCDX2 significantly synergizes with RAD51 to protect DNA against attack by the nucleases MRE11 and EXO1. DNA protection is contingent upon the ability of RAD51 to form a functional nucleoprotein filament on DNA. Collectively, our results provide evidence for a hitherto unknown function of RAD51 paralogs in synergizing with RAD51 nucleoprotein filament to prevent degradation of stressed replication forks.

Immunoprecipitation of RNA-DNA hybrid interacting proteins in Trypanosoma brucei reveals conserved and novel activities, including in the control of surface antigen expression needed for immune evasion by antigenic variation.

RNA-DNA hybrids are epigenetic features of genomes that provide a diverse and growing range of activities. Understanding of these functions has been informed by characterising the proteins that interact with the hybrids, but all such analyses have so far focused on mammals, meaning it is unclear if a similar spectrum of RNA-DNA hybrid interactors is found in other eukaryotes. The African trypanosome is a single-cell eukaryotic parasite of the Discoba grouping and displays substantial divergence in several aspects of core biology from its mammalian host. Here, we show that DNA-RNA hybrid immunoprecipitation coupled with mass spectrometry recovers 602 putative interactors in T. brucei mammal- and insect-infective cells, some providing activities also found in mammals and some lineage-specific. We demonstrate that loss of three factors, two putative helicases and a RAD51 paralogue, alters T. brucei nuclear RNA-DNA hybrid and DNA damage levels. Moreover, loss of each factor affects the operation of the parasite immune survival mechanism of antigenic variation. Thus, our work reveals the broad range of activities contributed by RNA-DNA hybrids to T. brucei biology, including new functions in host immune evasion as well as activities likely fundamental to eukaryotic genome function.

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

Homologous recombination (HR) is essential for error-free repair of DNA double-strand breaks, perturbed replication forks (RFs), and post-replicative single-stranded DNA (ssDNA) gaps. To initiate HR, the recombination mediator and tumor suppressor protein BRCA2 facilitates nucleation of RAD51 on ssDNA prior to stimulation of RAD51 filament growth by RAD51 paralogs. Although ssDNA binding by BRCA2 has been implicated in RAD51 nucleation, the function of double-stranded DNA (dsDNA) binding by BRCA2 remains unclear. Here, we exploit single-molecule (SM) imaging to visualize BRCA2-mediated RAD51 nucleation in real time using purified proteins. We report that BRCA2 nucleates and stabilizes RAD51 on ssDNA either directly or through an unappreciated diffusion-assisted delivery mechanism involving binding to and sliding along dsDNA, which requires the cooperative action of multiple dsDNA-binding modules in BRCA2. Collectively, our work reveals two distinct mechanisms of BRCA2-dependent RAD51 loading onto ssDNA, which we propose are critical for its diverse functions in maintaining genome stability and cancer suppression.

RAD51C-RAD51D interplays with MSH5 and regulates crossover maturation in rice meiosis.

Meiotic crossovers ensure accurate chromosome segregation and increase genetic diversity. RAD51C and RAD51D play an early role in facilitating RAD51 during homologous recombination. However, their later function in meiosis is largely unknown in plants. Here, through targeted disruption of RAD51C and RAD51D, we generated three new mutants and revealed their later meiotic role in crossover maturation. The rad51c-3 and rad51d-4 mutants showed a mixture of bivalents and univalents and no chromosomal entanglements, whereas rad51d-5 exhibited an intermediate phenotype with reduced chromosomal entanglements and increased bivalent formation compared with knockout alleles. Comparisons of RAD51 loadings and chromosomal entanglements in these single mutants, rad51c-3 rad51d-4, rad51c-3 dmc1a dmc1b, and rad51d-4 dmc1a dmc1b suggest that the retained level of RAD51 in mutants is required for uncovering their function in crossover formation. Reductions in chiasma frequency and later HEI10 foci in these mutants support that crossover maturation requires RAD51C and RAD51D. Moreover, interaction between RAD51D and MSH5 indicates that RAD51 paralogs may cooperate with MSH5 to ensure accurate Holliday junction processing into crossover products. This finding of the role of RAD51 paralogs in crossover control may be conserved from mammals to plants and advances our current understanding of these proteins.

Structure and function of the RAD51B-RAD51C-RAD51D-XRCC2 tumour suppressor.

Homologous recombination is a fundamental process of life. It is required for the protection and restart of broken replication forks, the repair of chromosome breaks and the exchange of genetic material during meiosis. Individuals with mutations in key recombination genes, such as BRCA2 (also known as FANCD1), or the RAD51 paralogues RAD51B, RAD51C (also known as FANCO), RAD51D, XRCC2 (also known as FANCU) and XRCC3,are predisposed to breast, ovarian and prostate cancers1-10 and the cancer-prone syndrome Fanconi anaemia11-13. The BRCA2 tumour suppressor protein-the product of BRCA2-is well characterized, but the cellular functions of the RAD51 paralogues remain unclear. Genetic knockouts display growth defects, reduced RAD51 focus formation, spontaneous chromosome abnormalities, sensitivity to PARP inhibitors and replication fork defects14,15, but the precise molecular roles of RAD51 paraloguesin fork stability, DNA repair and cancer avoidance remain unknown. Here we used cryo-electron microscopy, AlphaFold2 modelling and structural proteomics to determine the structure of the RAD51B-RAD51C-RAD51D-XRCC2 complex (BCDX2), revealing that RAD51C-RAD51D-XRCC2 mimics three RAD51 protomers aligned within a nucleoprotein filament, whereas RAD51B is highly dynamic. Biochemical and single-molecule analyses showed that BCDX2 stimulates the nucleation and extension of RAD51 filaments-which are essential for recombinational DNA repair-in reactions that depend on the coupled ATPase activities of RAD51B and RAD51C. Our studies demonstrate that BCDX2 orchestrates RAD51 assembly on single stranded DNA for replication fork protection and double strand break repair, in reactions that are critical for tumour avoidance.

The association of immune-related genes and the potential role of IL10 with biliary atresia.

Biliary atresia (BA) is a severe immune-related disease that is characterized by biliary obstruction and cholestasis. The etiology of BA is unclear, our aim was to explore the relationship between biliary tract inflammation and immune-related genes. We selected 14 SNPs in 13 immune-related genes and investigated their associations with BA by using a large case‒control cohort with a total of 503 cases and 1473 controls from southern China. SNP rs1518111 in interleukin10 (IL10) was identified as associated with BA (P = 5.79E-03; OR: 0.80; 95% CI: 0.68-0.94). The epistatic effects of the following pairwise interactions among these SNPs were associated with BA: signal transducer and activator of transcription 4 (STAT4) and chemokine (C-X-C motif) ligand 3 (CXCL3); STAT4 and damage-regulated autophagy modulator1 (DRAM1); CXCL3 and RAD51 paralog B (RAD51B); and interferon gamma (IFNG) and interleukin26 (IL26). Furthermore, we explored the potential role of IL-10 in the pathogenesis of the neonatal mouse model of BA. IL-10 effectively prevented biliary epithelial cell injury and biliary obstruction in murine BA as well as inhibit the activation of BA-related immune cells. In conclusion, this study provided strong evidence implicating IL10 as a susceptibility gene for BA in the southern Chinese population. This study provided strong evidence implicating IL10 as a susceptibility gene for BA in the southern Chinese population. This study could infer that IL-10 may play a protective role in BA mouse model. We found that four SNPs (rs7574865, rs352038, rs4622329, and rs4902562) have genetic interactions.

Expression profiles and potential roles of serum tRNA‑derived fragments in diabetic nephropathy.

Diabetic nephropathy (DN) is one of the most important causes of end-stage renal disease and current treatments are ineffective in preventing its progression. Transfer RNA (tRNA)-derived fragments (tRFs), which are small non-coding fragments derived from tRNA precursors or mature tRNAs, have a critical role in various human diseases. The present study aimed to investigate the expression profile and potential functions of tRFs in DN. High-throughput sequencing technology was employed to detect the differential serum levels of tRFs between DN and diabetes mellitus and to validate the reliability of the sequencing results using reverse transcription-quantitative PCR. Ultimately, six differentially expressed (DE) tRFs were identified (P<0.05; |log2fold change| ≥1), including three upregulated (tRF5-GluCTC, tRF5-AlaCGC and tRF5-ValCAC) and three downregulated tRFs (tRF5-GlyCCC, tRF3-GlyGCC and tRF3-IleAAT). Potential functions and regulatory mechanisms of these DE tRFs were further evaluated using an applied bioinformatics-based analysis. Gene ontology analysis revealed that the DE tRFs are mainly enriched in biological processes, including axon guidance, Rad51 paralog (Rad51)B-Rad51C-Rad51D-X-Ray repair cross-complementing 2 complex, nuclear factor of activated T-cells protein binding and fibroblast growth factor-activated receptor activity. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that they are associated with axon guidance, neurotrophin signaling, mTOR signaling, AMPK signaling and epidermal growth factor receptor family signaling pathways. In conclusion, the present findings indicated that tRFs were DE in DN and may be involved in the regulation of DN pathology through multiple pathways, thereby providing a new perspective for the study of DN therapeutic targets.

Rad51 filaments assembled in the absence of the complex formed by the Rad51 paralogs Rad55 and Rad57 are outcompeted by translesion DNA polymerases on UV-induced ssDNA gaps.

The bypass of DNA lesions that block replicative polymerases during DNA replication relies on DNA damage tolerance pathways. The error-prone translesion synthesis (TLS) pathway depends on specialized DNA polymerases that incorporate nucleotides in front of base lesions, potentially inducing mutagenesis. Two error-free pathways can bypass the lesions: the template switching pathway, which uses the sister chromatid as a template, and the homologous recombination pathway (HR), which also can use the homologous chromosome as template. The balance between error-prone and error-free pathways controls the mutagenesis level. Therefore, it is crucial to precisely characterize factors that influence the pathway choice to better understand genetic stability at replication forks. In yeast, the complex formed by the Rad51 paralogs Rad55 and Rad57 promotes HR and template-switching at stalled replication forks. At DNA double-strand breaks (DSBs), this complex promotes Rad51 filament formation and stability, notably by counteracting the Srs2 anti-recombinase. To explore the role of the Rad55-Rad57 complex in error-free pathways, we monitored the genetic interactions between Rad55-Rad57, the translesion polymerases Polζ or Polη, and Srs2 following UV radiation that induces mostly single-strand DNA gaps. We found that the Rad55-Rad57 complex was involved in three ways. First, it protects Rad51 filaments from Srs2, as it does at DSBs. Second, it promotes Rad51 filament stability independently of Srs2. Finally, we observed that UV-induced HR is almost abolished in Rad55-Rad57 deficient cells, and is partially restored upon Polζ or Polη depletion. Hence, we propose that the Rad55-Rad57 complex is essential to promote Rad51 filament stability on single-strand DNA gaps, notably to counteract the error-prone TLS polymerases and mutagenesis.

RAD51 and RAD51B Play Diverse Roles in the Repair of DNA Double Strand Breaks in Physcomitrium patens.

RAD51 is involved in finding and invading homologous DNA sequences for accurate homologous recombination (HR). Its paralogs have evolved to regulate and promote RAD51 functions. The efficient gene targeting and high HR rates are unique in plants only in the moss Physcomitrium patens (P. patens). In addition to two functionally equivalent RAD51 genes (RAD1-1 and RAD51-2), other RAD51 paralogues were also identified in P. patens. For elucidation of RAD51's involvement during DSB repair, two knockout lines were constructed, one mutated in both RAD51 genes (Pprad51-1-2) and the second with mutated RAD51B gene (Pprad51B). Both lines are equally hypersensitive to bleomycin, in contrast to their very different DSB repair efficiency. Whereas DSB repair in Pprad51-1-2 is even faster than in WT, in Pprad51B, it is slow, particularly during the second phase of repair kinetic. We interpret these results as PpRAD51-1 and -2 being true functional homologs of ancestral RAD51 involved in the homology search during HR. Absence of RAD51 redirects DSB repair to the fast NHEJ pathway and leads to a reduced 5S and 18S rDNA copy number. The exact role of the RAD51B paralog remains unclear, though it is important in damage recognition and orchestrating HR response.

Genetic variants in African-American and Hispanic patients with breast cancer.

Breast cancer is a disease with significant health disparity affecting mortality in minority women. The present study examined the genetic makeup of breast cancers in African-American and Hispanic/Latinx patients to determine specific genetic mutations associated with breast cancer in the minority population from South Los Angeles, United States. Whole-exome sequencing was performed on DNA extracted from breast cancer tumor biopsies collected from 13 African-American and 15 Hispanic women and 8 matched-normal samples for each ethnic category. The results were analyzed using Ensemble Variant Effect Predictor and Mutation Significance. Additionally, a comparative analysis with The Cancer Genome Atlas data was provided. Our data revealed somatic mutations in genes such as SET domain containing (lysine methyltransferase) 8, serine protease 1 and AT-rich interaction domain 1B (ARID1B) and known breast cancer genes, such as BRCA1/2, TP53 and the DNA damage response genes across all ethnicities. Additionally, Hispanic patients had BRCA1 associated RING domain 1B (BARD1) variants, while African-American patients had higher numbers of nonsynonymous variants in the RAD51 paralog B (RAD51B), ARID1B and X-ray repair cross complementing 3 (XRCC3) genes. In addition, our patients exhibited mutational signature enrichment that indicated DNA homologous recombination repair deficiencies. Therefore, African-American and Hispanic breast cancer samples showed considerable overlap in breast cancer genetic mutations. However, there are differences in specific genetic variants in TP53, BRCA1/2, BARD1 or ARID1B, which will require further study of their role in tumorigenesis.

Systematic proximal mapping of the classical RAD51 paralogs unravel functionally and clinically relevant interactors for genome stability.

Homologous recombination (HR) plays an essential role in the maintenance of genome stability by promoting the repair of cytotoxic DNA double strand breaks (DSBs). More recently, the HR pathway has emerged as a core component of the response to replication stress, in part by protecting stalled replication forks from nucleolytic degradation. In that regard, the mammalian RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) have been involved in both HR-mediated DNA repair and collapsed replication fork resolution. Still, it remains largely obscure how they participate in both processes, thereby maintaining genome stability and preventing cancer development. To gain better insight into their contribution in cellulo, we mapped the proximal interactome of the classical RAD51 paralogs using the BioID approach. Aside from identifying the well-established BCDX2 and CX3 sub-complexes, the spliceosome machinery emerged as an integral component of our proximal mapping, suggesting a crosstalk between this pathway and the RAD51 paralogs. Furthermore, we noticed that factors involved RNA metabolic pathways are significantly modulated within the BioID of the classical RAD51 paralogs upon exposure to hydroxyurea (HU), pointing towards a direct contribution of RNA processing during replication stress. Importantly, several members of these pathways have prognostic potential in breast cancer (BC), where their RNA expression correlates with poorer patient outcome. Collectively, this study uncovers novel functionally relevant partners of the different RAD51 paralogs in the maintenance of genome stability that could be used as biomarkers for the prognosis of BC.

Homologous recombination–deficient mutation cluster in tumor suppressor RAD51C identified by comprehensive analysis of cancer variants

Mutations in homologous recombination (HR) genes, including BRCA1, BRCA2, and the RAD51 paralog RAD51C, predispose to tumorigenesis and sensitize cancers to DNA-damaging agents and poly(ADP ribose) polymerase inhibitors. However, ∼800 missense variants of unknown significance have been identified for RAD51C alone, impairing cancer risk assessment and therapeutic strategies. Here, we interrogated >50 RAD51C missense variants, finding that mutations in residues conserved with RAD51 strongly predicted HR deficiency and disrupted interactions with other RAD51 paralogs. A cluster of mutations was identified in and around the Walker A box that led to impairments in HR, interactions with three other RAD51 paralogs, binding to single-stranded DNA, and ATP hydrolysis. We generated structural models of the two RAD51 paralog complexes containing RAD51C, RAD51B-RAD51C-RAD51D-XRCC2 and RAD51C-XRCC3. Together with our functional and biochemical analyses, the structural models predict ATP binding at the interface of RAD51C interactions with other RAD51 paralogs, similar to interactions between monomers in RAD51 filaments, and explain the failure of RAD51C variants in binding multiple paralogs. Ovarian cancer patients with variants in this cluster showed exceptionally long survival, which may be relevant to the reversion potential of the variants. This comprehensive analysis provides a framework for RAD51C variant classification. Importantly, it also provides insight into the functioning of the RAD51 paralog complexes.

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.

Guiding ATR and PARP inhibitor combinationswith chemogenomic screens.

Combinations of ataxia telangiectasia- and Rad3-related kinase inhibitors (ATRis) and poly(ADP-ribose) polymerase inhibitors (PARPis) synergistically kill tumor cells through modulation of complementary DNA repair pathways, but their tolerability is limited by hematological toxicities. To address this, we performed a genome-wide CRISPR-Cas9 screen to identify genetic alterations that hypersensitize cells to a combination of the ATRi RP-3500 with PARPi, including deficiency in RNase H2, RAD51 paralog mutations, or the "alternative lengthening of telomeres" telomere maintenance mechanism. We show that RP-3500 and PARPi combinations kill cells carrying these genetic alterations at doses sub-therapeutic as single agents. We also demonstrate the mechanism of combination hypersensitivity in RNase H2-deficient cells, where we observe an irreversible replication catastrophe, allowing us to design a highly efficacious and tolerable invivo dosing schedule. We present a comprehensive dataset to inform development of ATRi and PARPi combinations and an experimental framework applicable to other drug combination strategies.

The Association Between Breast Cancer and Blood-Based Methylation of CD160, ISYNA1 and RAD51B in the Chinese Population.

Recent studies have identified DNA methylation signatures in the white blood cells as potential biomarkers for breast cancer (BC) in the European population. Here, we investigated the association between BC and blood-based methylation of cluster of differentiation 160 (CD160), inositol-3-phosphate synthase 1 (ISYNA1) and RAD51 paralog B (RAD51B) genes in the Chinese population. Peripheral blood samples were collected from two independent case-control studies with a total of 272 sporadic early-stage BC cases (76.5% at stage I&II) and 272 cancer-free female controls. Mass spectrometry was applied to quantitatively measure the levels of DNA methylation. The logistic regression and non-parametric tests were used for the statistical analyses. In contrast to the protective effects reported in European women, we reported the blood-based hypomethylation in CD160, ISYNA1 and RAD51B as risk factors for BC in the Chinese population (CD160_CpG_3, CD160_CpG_4/cg20975414, ISYNA1_CpG_2, RAD51B_CpG_3 and RAD51B_CpG_4; odds ratios (ORs) per -10% methylation ranging from 1.08 to 1.67, p < 0.05 for all). Moreover, hypomethylation of CD160, ISYNA1 and RAD51B was significantly correlated with age, BC subtypes including estrogen receptor (ER)-negative BC tumors, triple negative tumors, BC cases with larger size, advanced stages and more lymph node involvement. Our results supported the report in European women that BC is associated with altered methylation of CD160, ISYNA1 and RAD51B in the peripheral blood, although the effects are opposite in the Chinese population. The difference between the two populations may be due to variant genetic background or life styles, implicating that the validations of epigenetic biomarkers in variant ethnic groups are warranted.