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 and SWS1. 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 SWS1 complex formation. Together, we show that SWSAP1-SWS1 stimulates RAD51-dependent high-fidelity repair and may be an important new cancer therapeutic target.

Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair.

Nucleotide excision repair (NER) promotes genomic integrity by removing bulky DNA adducts introduced by external factors such as ultraviolet light. Defects in NER enzymes are associated with pathological conditions such as Xeroderma Pigmentosum, trichothiodystrophy, and Cockayne syndrome. A critical step in NER is the binding of the Xeroderma Pigmentosum group A protein (XPA) to the ss/ds DNA junction. To better capture the dynamics of XPA interactions with DNA during NER we have utilized the fluorescence enhancement through non-canonical amino acids (FEncAA) approach. 4-azido-L-phenylalanine (4AZP or pAzF) was incorporated at Arg-158 in human XPA and conjugated to Cy3 using strain-promoted azide-alkyne cycloaddition. The resulting fluorescent XPA protein (XPACy3) shows no loss in DNA binding activity and generates a robust change in fluorescence upon binding to DNA. Here we describe methods to generate XPACy3 and detail in vitro experimental conditions required to stably maintain the protein during biochemical and biophysical studies.

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.

Human hnRNPA1 reorganizes telomere-bound Replication Protein A.

Human replication protein A (RPA) is a heterotrimeric ssDNA binding protein responsible for many aspects of cellular DNA metabolism. Dynamic interactions of the four RPA DNA binding domains (DBDs) with DNA control replacement of RPA by downstream proteins in various cellular metabolic pathways. RPA plays several important functions at telomeres where it binds to and melts telomeric G-quadruplexes, non-canonical DNA structures formed at the G-rich telomeric ssDNA overhangs. Here, we combine single-molecule total internal reflection fluorescence microscopy (smTIRFM) and mass photometry (MP) with biophysical and biochemical analyses to demonstrate that heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) specifically remodels RPA bound to telomeric ssDNA by dampening the RPA configurational dynamics and forming a ternary complex. Uniquely, among hnRNPA1 target RNAs, telomeric repeat-containing RNA (TERRA) is selectively capable of releasing hnRNPA1 from the RPA-telomeric DNA complex. We speculate that this telomere specific RPA-DNA-hnRNPA1 complex is an important structure in telomere protection. One Sentence Summary: At the single-stranded ends of human telomeres, the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) binds to and modulates conformational dynamics of the ssDNA binding protein RPA forming a ternary complex which is controlled by telomeric repeat-containing RNA (TERRA).

Human RAD52 double-ring remodels replication forks restricting fork reversal.

Human RAD52 1,2 is a multifunctional DNA repair protein involved in several cellular events that support genome stability including protection of stalled DNA replication forks from excessive degradation 3-7 . In its gatekeeper role, RAD52 binds to and stabilizes stalled replication forks during replication stress protecting them from reversal by SMARCAL1 5 . The structural and molecular mechanism of the RAD52-mediated fork protection remains elusive. Here, using P1 nuclease sensitivity, biochemical and single-molecule analyses we show that RAD52 dynamically remodels replication forks through its strand exchange activity. The presence of the ssDNA binding protein RPA at the fork modulates the kinetics of the strand exchange without impeding the reaction outcome. Mass photometry and single-particle cryo-electron microscopy show that the replication fork promotes a unique nucleoprotein structure containing head-to-head arrangement of two undecameric RAD52 rings with an extended positively charged surface that accommodates all three arms of the replication fork. We propose that the formation and continuity of this surface is important for the strand exchange reaction and for competition with SMARCAL1.

Depletion of Labile Iron Induces Replication Stress and Enhances Responses to Chemoradiation in Non-Small-Cell Lung Cancer.

The intracellular redox-active labile iron pool (LIP) is weakly chelated and available for integration into the iron metalloproteins that are involved in diverse cellular processes, including cancer cell-specific metabolic oxidative stress. Abnormal iron metabolism and elevated LIP levels are linked to the poor survival of lung cancer patients, yet the underlying mechanisms remain unclear. Depletion of the LIP in non-small-cell lung cancer cell lines using the doxycycline-inducible overexpression of the ferritin heavy chain (Ft-H) (H1299 and H292), or treatment with deferoxamine (DFO) (H1299 and A549), inhibited cell growth and decreased clonogenic survival. The Ft-H overexpression-induced inhibition of H1299 and H292 cell growth was also accompanied by a significant delay in transit through the S-phase. In addition, both Ft-H overexpression and DFO in H1299 resulted in increased single- and double-strand DNA breaks, supporting the involvement of replication stress in the response to LIP depletion. The Ft-H and DFO treatment also sensitized H1299 to VE-821, an inhibitor of ataxia telangiectasis and Rad2-related (ATR) kinase, highlighting the potential of LIP depletion, combined with DNA damage response modifiers, to alter lung cancer cell responses. In contrast, only DFO treatment effectively reduced the LIP, clonogenic survival, cell growth, and sensitivity to VE-821 in A549 non-small-cell lung cancer cells. Importantly, the Ft-H and DFO sensitized both H1299 and A549 to chemoradiation in vitro, and Ft-H overexpression increased the efficacy of chemoradiation in vivo in H1299. These results support the hypothesis that the depletion of the LIP can induce genomic instability, cell death, and potentiate therapeutic responses to chemoradiation in NSCLC.

Antagonistic roles of canonical and Alternative-RPA in disease-associated tandem CAG repeat instability.

Expansions of repeat DNA tracts cause >70 diseases, and ongoing expansions in brains exacerbate disease. During expansion mutations, single-stranded DNAs (ssDNAs) form slipped-DNAs. We find the ssDNA-binding complexes canonical replication protein A (RPA1, RPA2, and RPA3) and Alternative-RPA (RPA1, RPA3, and primate-specific RPA4) are upregulated in Huntington disease and spinocerebellar ataxia type 1 (SCA1) patient brains. Protein interactomes of RPA and Alt-RPA reveal unique and shared partners, including modifiers of CAG instability and disease presentation. RPA enhances in vitro melting, FAN1 excision, and repair of slipped-CAGs and protects against CAG expansions in human cells. RPA overexpression in SCA1 mouse brains ablates expansions, coincident with decreased ATXN1 aggregation, reduced brain DNA damage, improved neuron morphology, and rescued motor phenotypes. In contrast, Alt-RPA inhibits melting, FAN1 excision, and repair of slipped-CAGs and promotes CAG expansions. These findings suggest a functional interplay between the two RPAs where Alt-RPA may antagonistically offset RPA's suppression of disease-associated repeat expansions, which may extend to other DNA processes.

Germline landscape of RPA1, RPA2 and RPA3 variants in pediatric malignancies: identification of RPA1 as a novel cancer predisposition candidate gene.

Replication Protein A (RPA) is single-strand DNA binding protein that plays a key role in the replication and repair of DNA. RPA is a heterotrimer made of 3 subunits - RPA1, RPA2, and RPA3. Germline pathogenic variants affecting RPA1 were recently described in patients with Telomere Biology Disorders (TBD), also known as dyskeratosis congenita or short telomere syndrome. Premature telomere shortening is a hallmark of TBD and results in bone marrow failure and predisposition to hematologic malignancies. Building on the finding that somatic mutations in RPA subunit genes occur in ~1% of cancers, we hypothesized that germline RPA alterations might be enriched in human cancers. Because germline RPA1 mutations are linked to early onset TBD with predisposition to myelodysplastic syndromes, we interrogated pediatric cancer cohorts to define the prevalence and spectrum of rare/novel and putative damaging germline RPA1, RPA2, and RPA3 variants. In this study of 5,993 children with cancer, 75 (1.25%) harbored heterozygous rare (non-cancer population allele frequency (AF) < 0.1%) variants in the RPA heterotrimer genes, of which 51 cases (0.85%) had ultra-rare (AF < 0.005%) or novel variants. Compared with Genome Aggregation Database (gnomAD) non-cancer controls, there was significant enrichment of ultra-rare and novel RPA1, but not RPA2 or RPA3, germline variants in our cohort (adjusted p-value < 0.05). Taken together, these findings suggest that germline putative damaging variants affecting RPA1 are found in excess in children with cancer, warranting further investigation into the functional role of these variants in oncogenesis.

Contrasting roles of different mismatch repair proteins in basal-like breast cancer.

The mismatch repair (MMR) pathway is known as a tumor suppressive pathway and genes involved in MMR are commonly mutated in hereditary colorectal or other cancer types. However, the function of MMR genes/proteins in breast cancer progression and metastasis are largely unknown. We found that MSH2, but not MLH1, is highly enriched in basal-like breast cancer (BLBC) and that its protein expression is inversely correlated with overall survival time (OS). MSH2 expression is frequently elevated due to genomic amplification or gain-of-expression in BLBC, which results in increased MSH2 protein to pair with MSH6 (collectively referred to as MutSα). Genetic deletion of MSH2 or MLH1 results in a contrasting phenotype in metastasis, with MSH2-deletion leading to reduced metastasis and MLH1-deletion to enhanced liver or lung metastasis. Mechanistically, MSH2-deletion induces the expression of a panel of chemokines in BLBC via epigenetic and/or transcriptional regulation, which leads to an immune reactive tumor microenvironment (TME) and elevated immune cell infiltrations. MLH1 is not correlated with chemokine expression and/or immune cell infiltration in BLBC, but its deletion results in strong accumulation of neutrophils that are known for metastasis promotion. Our study supports the differential functions of MSH2 and MLH1 in BLBC progression and metastasis, which challenges the paradigm of the MMR pathway as a universal tumor suppressive mechanism.

Small molecule nitroalkenes inhibit RAD51-mediated homologous recombination and amplify triple-negative breast cancer cell killing by DNA-directed therapies.

Nitro fatty acids (NO2-FAs) are endogenously generated lipid signaling mediators from metabolic and inflammatory reactions between conjugated diene fatty acids and nitric oxide or nitrite-derived reactive species. NO2-FAs undergo reversible Michael addition with hyperreactive protein cysteine thiolates to induce posttranslational protein modifications that can impact protein function. Herein, we report a novel mechanism of action of natural and non-natural nitroalkenes structurally similar to (E) 10-nitro-octadec-9-enoic acid (CP-6), recently de-risked by preclinical Investigational New Drug-enabling studies and Phase 1 and Phase 2 clinical trials and found to induce DNA damage in a TNBC xenograft by inhibiting homologous-recombination (HR)-mediated repair of DNA double-strand breaks (DSB). CP-6 specifically targets Cys319, essential in RAD51-controlled HR-mediated DNA DSB repair in cells. A nitroalkene library screen identified two structurally different nitroalkenes, a non-natural fatty acid [(E) 8-nitro-nonadec-7-enoic acid (CP-8)] and a dicarboxylate ester [dimethyl (E)nitro-oct-4-enedioate (CP-23)] superior to CP-6 in TNBC cells killing, synergism with three different inhibitors of the poly ADP-ribose polymerase (PARP) and γ-IR. CP-8 and CP-23 effectively inhibited γ-IR-induced RAD51 foci formation and HR in a GFP-reported assay but did not affect benign human epithelial cells or cell cycle phases. In vivo, CP-8 and CP-23's efficacies diverged as only CP-8 showed promising anticancer activities alone and combined with the PARP inhibitor talazoparib in an HR-proficient TNBC mouse model. As preliminary preclinical toxicology analysis also suggests CP-8 as safe, our data endorse CP-8 as a novel anticancer molecule for treating cancers sensitive to homologous recombination-mediated DNA repair inhibitors.

Small molecule nitroalkenes inhibit RAD51-mediated homologous recombination and amplify triple-negative breast cancer cell killing by DNA-directed therapies.

Nitro fatty acids (NO 2 -FAs) are endogenously generated lipid signaling mediators from metabolic and inflammatory reactions between conjugated diene fatty acids and nitric oxide or nitrite-derived reactive species. NO 2 -FAs undergo reversible Michael addition with hyperreactive protein cysteine thiolates to induce posttranslational protein modifications that can impact protein function. Herein, we report a novel mechanism of action of natural and non-natural nitroalkenes structurally similar to ( E ) 10-nitro-octadec-9-enoic acid (CP-6), recently de-risked by preclinical Investigational New Drug-enabling studies and Phase 1 and Phase 2 clinical trials and found to induce DNA damage in a TNBC xenograft by inhibiting homologous-recombination (HR)-mediated repair of DNA double-strand breaks (DSB). CP-6 specifically targets Cys319, essential in RAD51-controlled HR-mediated DNA DSB repair in cells. A nitroalkene library screen identified two structurally different nitroalkenes, a non-natural fatty acid [( E ) 8-nitro- nonadec-7-enoic acid (CP-8)] and a dicarboxylate ester [dimethyl ( E )nitro-oct-4-enedioate (CP- 23)] superior to CP-6 in TNBC cells killing, synergism with three different inhibitors of the poly ADP-ribose polymerase (PARP) and γ-IR. CP-8 and CP-23 effectively inhibited γ-IR-induced RAD51 foci formation and HR in a GFP-reported assay but did not affect benign human epithelial cells or cell cycle phases. In vivo, CP-8 and CP-23's efficacies diverged as only CP-8 showed promising anticancer activities alone and combined with the PARP inhibitor talazoparib in an HR-proficient TNBC mouse model. As preliminary preclinical toxicology analysis also suggests CP-8 as safe, our data endorse CP-8 as a novel anticancer molecule for treating cancers sensitive to homologous recombination-mediated DNA repair inhibitors.

Single-Molecule Analysis of the Improved Variants of the G-Quadruplex Recognition Protein G4P.

As many as 700,000 unique sequences in the human genome are predicted to fold into G-quadruplexes (G4s), non-canonical structures formed by Hoogsteen guanine-guanine pairing within G-rich nucleic acids. G4s play both physiological and pathological roles in many vital cellular processes including DNA replication, DNA repair and RNA transcription. Several reagents have been developed to visualize G4s in vitro and in cells. Recently, Zhen et al. synthesized a small protein G4P based on the G4 recognition motif from RHAU (DHX36) helicase (RHAU specific motif, RSM). G4P was reported to bind the G4 structures in cells and in vitro, and to display better selectivity toward G4s than the previously published BG4 antibody. To get insight into G4P- G4 interaction kinetics and selectivity, we purified G4P and its expanded variants, and analyzed their G4 binding using single-molecule total internal reflection fluorescence microscopy and mass photometry. We found that G4P binds to various G4s with affinities defined mostly by the association rate. Doubling the number of the RSM units in the G4P increases the protein's affinity for telomeric G4s and its ability to interact with sequences folding into multiple G4s.

Therapeutic disruption of RAD52-ssDNA complexation via novel drug-like inhibitors.

RAD52 protein is a coveted target for anticancer drug discovery. Similar to poly-ADP-ribose polymerase (PARP) inhibitors, pharmacological inhibition of RAD52 is synthetically lethal with defects in genome caretakers BRCA1 and BRCA2 (∼25% of breast and ovarian cancers). Emerging structure activity relationships for RAD52 are complex, making it challenging to transform previously identified disruptors of the RAD52-ssDNA interaction into drug-like leads using traditional medicinal chemistry approaches. Using pharmacophoric informatics on the RAD52 complexation by epigallocatechin (EGC), and the Enamine in silico REAL database, we identified six distinct chemical scaffolds that occupy the same physical space on RAD52 as EGC. All six were RAD52 inhibitors (IC50 ∼23-1200 µM) with two of the compounds (Z56 and Z99) selectively killing BRCA-mutant cells and inhibiting cellular activities of RAD52 at micromolar inhibitor concentrations. While Z56 had no effect on the ssDNA-binding protein RPA and was toxic to BRCA-mutant cells only, Z99 inhibited both proteins and displayed toxicity towards BRCA-complemented cells. Optimization of the Z99 scaffold resulted in a set of more powerful and selective inhibitors (IC50 ∼1.3-8 µM), which were only toxic to BRCA-mutant cells. RAD52 complexation by Z56, Z99 and its more specific derivatives provide a roadmap for next generation of cancer therapeutics.

Single-molecule analysis of the improved variants of the G-quadruplex recognition protein G4P.

As many as 700,000 unique sequences in the human genome are predicted to fold into G-quadruplexes (G4s), non-canonical structures formed by Hoogsteen guanine-guanine pairing within G-rich nucleic acids. G4s play both physiological and pathological roles in many vital cellular processes including DNA replication, DNA repair and RNA transcription. Several reagents have been developed to visualize G4s in vitro and in cells. Recently, Zhen et al . synthesized a small protein G4P based on the G4 recognition motif from RHAU (DHX36) helicase (RHAU specific motif, RSM). G4P was reported to bind the G4 structures in cells and in vitro , and to display better selectivity towards G4s than the previously published BG4 antibody. To get insight into the G4P-G4 interaction kinetics and selectivity, we purified G4P and its expanded variants, and analyzed their G4 binding using single-molecule total internal reflection fluorescence microscopy and mass photometry. We found that G4P binds to various G4s with affinities defined mostly by the association rate. Doubling the number of the RSM units in the G4P increases the protein's affinity for telomeric G4s and its ability to interact with sequences folding into multiple G4s.

Visualizing the coordination of apurinic/apyrimidinic endonuclease (APE1) and DNA polymerase ß during base excision repair.

Base excision repair (BER) is carried out by a series of proteins that function in a step-by-step process to identify, remove, and replace DNA damage. During BER, the DNA transitions through various intermediate states as it is processed by each DNA repair enzyme. Left unrepaired, these BER intermediates can transition into double-stranded DNA breaks and promote genome instability. Previous studies have proposed a short-lived complex consisting of the BER intermediate, the incoming enzyme, and the outgoing enzyme at each step of the BER pathway to protect the BER intermediate. The transfer of BER intermediates between enzymes, known as BER coordination or substrate channeling, remains poorly understood. Here, we utilize single-molecule total internal reflection fluorescence microscopy to investigate the mechanism of BER coordination between apurinic/apyrimidinic endonuclease 1 (APE1) and DNA polymerase ß (Pol ß). When preformed complexes of APE1 and the incised abasic site product (APE1 product and Pol ß substrate) were subsequently bound by Pol ß, the Pol ß enzyme dissociated shortly after binding in most of the observations. In the events where Pol ß binding was followed by APE1 dissociation during substrate channeling, Pol ß remained bound for a longer period of time to allow disassociation of APE1. Our results indicate that transfer of the BER intermediate from APE1 to Pol ß during BER is dependent on the dissociation kinetics of APE1 and the duration of the ternary complex on the incised abasic site.

Fork-Remodeling Helicase Rad5 Preferentially Reverses Replication Forks with Gaps in the Leading Strand.

DNA damage bypass pathways promote the replication of damaged DNA when replication forks stall at sites of DNA damage. Template switching is a DNA damage bypass pathway in which fork-reversal helicases convert stalled replication forks into four-way DNA junctions called chicken foot intermediates, which are subsequently extended by replicative DNA polymerases. In yeast, fork-reversal is carried out by the Rad5 helicase using an unknown mechanism. To better understand the mechanism of Rad5 and its specificity for different fork DNA substrates, we used a FRET-based assay to observe fork reversal in real time. We examined the ability of Rad5 to bind and catalyze the reversal of various fork DNA substrates in the presence of short gaps in the leading or lagging strand as well as in the presence or absence of RPA and RNA primers in the lagging strand. We found that Rad5 preferentially reverses fork DNA substrates with short gaps (10 to 30 nt.) in the leading strand. Thus, Rad5 preferentially reverses fork DNA substrates that form chicken foot intermediates with 5' overhangs that can be extended by replicative DNA polymerases during the subsequent steps of template switching.

A moving target for drug discovery: Structure activity relationship and many genome (de)stabilizing functions of the RAD52 protein.

BRCA-ness phenotype, a signature of many breast and ovarian cancers, manifests as deficiency in homologous recombination, and as defects in protection and repair of damaged DNA replication forks. A dependence of such cancers on DNA repair factors less important for survival of BRCA-proficient cells, offers opportunities for development of novel chemotherapeutic interventions. The first drugs targeting BRCA-deficient cancers, poly-ADP-ribose polymerase (PARP) inhibitors have been approved for the treatment of advanced, chemotherapy resistant cancers in patients with BRCA1/2 germline mutations. Nine additional proteins that can be targeted to selectively kill BRCA-deficient cancer cells have been identified. Among them, a DNA repair protein RAD52 is an especially attractive target due to general tolerance of the RAD52 loss of function, and protective role of an inactivating mutation. Yet, the effective pharmacological inhibitors of RAD52 have not been forthcoming. In this review, we discuss advances in the state of our knowledge of the RAD52 structure, activities and cellular functions, with a specific focus on the features that make RAD52 an attractive, but difficult drug target.

Redox regulation of RAD51 Cys319 and homologous recombination by peroxiredoxin 1.

RAD51 is a critical recombinase that functions in concert with auxiliary mediator proteins to direct the homologous recombination (HR) DNA repair pathway. We show that Cys319 RAD51 possesses nucleophilic characteristics and is important for irradiation-induced RAD51 foci formation and resistance to inhibitors of poly (ADP-ribose) polymerase (PARP). We have previously identified that cysteine (Cys) oxidation of proteins can be important for activity and modulated via binding to peroxiredoxin 1 (PRDX1). PRDX1 reduces peroxides and coordinates the signaling actions of protein binding partners. Loss of PRDX1 inhibits irradiation-induced RAD51 foci formation and represses HR DNA repair. PRDX1-deficient human breast cancer cells and mouse embryonic fibroblasts display disrupted RAD51 foci formation and decreased HR, resulting in increased DNA damage and sensitization of cells to irradiation. Following irradiation cells deficient in PRDX1 had increased incorporation of the sulfenylation probe DAz-2 in RAD51 Cys319, a functionally-significant, thiol that PRDX1 is critical for maintaining in a reduced state. Molecular dynamics (MD) simulations of dT-DNA bound to a non-oxidized RAD51 protein showed tight binding throughout the simulation, while dT-DNA dissociated from an oxidized Cys319 RAD51 filament. These novel data establish RAD51 Cys319 as a functionally-significant site for the redox regulation of HR and cellular responses to IR.

Gain-of-function mutations in RPA1 cause a syndrome with short telomeres and somatic genetic rescue.

Human telomere biology disorders (TBD)/short telomere syndromes (STS) are heterogeneous disorders caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify 3 germline heterozygous missense variants in the RPA1 gene in 4 unrelated probands presenting with short telomeres and varying clinical features of TBD/STS, including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, or skin manifestations. All variants cluster to DNA-binding domain A of RPA1 protein. RPA1 is a single-strand DNA-binding protein required for DNA replication and repair and involved in telomere maintenance. We showed that RPA1E240K and RPA1V227A proteins exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function, whereas RPA1T270A has binding properties similar to wild-type protein. To study the mutational effect in a cellular system, CRISPR/Cas9 was used to knock-in the RPA1E240K mutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patients with RPA1E240K, we discovered somatic genetic rescue in hematopoietic cells due to an acquired truncating cis RPA1 mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the 2 somatic genetic rescue events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1 variants in individuals with TBD/STS.