Enhanced recombination empowers the detection and mapping of Quantitative Trait Loci.

Modern plant breeding, such as genomic selection and gene editing, is based on the knowledge of the genetic architecture of desired traits. Quantitative trait loci (QTL) analysis, which combines high throughput phenotyping and genotyping of segregating populations, is a powerful tool to identify these genetic determinants and to decipher the underlying mechanisms. However, meiotic recombination, which shuffles genetic information between generations, is limited: Typically only one to two exchange points, called crossovers, occur between a pair of homologous chromosomes. Here we test the effect on QTL analysis of boosting recombination, by mutating the anti-crossover factors RECQ4 and FIGL1 in Arabidopsis thaliana full hybrids and lines in which a single chromosome is hybrid. We show that increasing recombination ~6-fold empowers the detection and resolution of QTLs, reaching the gene scale with only a few hundred plants. Further, enhanced recombination unmasks some secondary QTLs undetected under normal recombination. These results show the benefits of enhanced recombination to decipher the genetic bases of traits.

James German and the Quest to Understand Human RECQ Helicase Deficiencies.

James German's work to establish the natural history and cancer risk associated with Bloom syndrome (BS) has had a strong influence on the generation of scientists and clinicians working to understand other RECQ deficiencies and heritable cancer predisposition syndromes. I summarize work by us and others below, inspired by James German's precedents with BS, to understand and compare BS with the other heritable RECQ deficiency syndromes with a focus on Werner syndrome (WS). What we know, unanswered questions and new opportunities are discussed, as are potential ways to treat or modify WS-associated disease mechanisms and pathways.

ATM, BLM, and CDH1 gene co-mutations in a high-grade endometrial stromal sarcoma patient with multiple abdominal cavity metastases: a case report and literature review.

BACKGROUND: High-grade endometrial stromal sarcoma (HG-ESS) is a rare malignant tumor with poor prognosis. To overcome the limitations of current treatment for advanced patients, the intervention of targeted drug therapy is urgently needed. CASE PRESENTATION: A 74-year-old married woman who presented with abdominal distension and lower abdominal pain was admitted to Hebei General Hospital. After surgery, immunohistochemical staining revealed a malignant tumor which was consistent with HG-ESS. Tumor recurrence occurred 2 months after surgery. Then the patient underwent chemotherapy with two courses but responded poorly. Subsequently we observed ATM, BLM, and CDH1 co-mutations by Next Generation Sequencing (NGS). Then the patient received pamiparib, which resulted in a 10-month progression-free survival (PFS) and is now stable with the administration of sintilimab in combination with pamiparib and anlotinib. CONCLUSIONS: Due to the successful use of poly ADP-ribose polymerase inhibitor (PARPi) on HG-ESS, we suggest that the selection of effective targeted drugs combined with anti- programmed death-1 (PD-1) drug therapy based on genetic testing may become a new option for the treatment of homologous repair deficient (HR-deficient) HG-ESS.

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.

Bloom syndrome DNA helicase mitigates mismatch repair-dependent apoptosis.

Generation of O6-methylguanine (O6-meG) by DNA-alkylating agents such as N-methyl N-nitrosourea (MNU) activates the multiprotein mismatch repair (MMR) complex and the checkpoint response involving ATR/CHK1 and ATM/CHK2 kinases, which may in turn trigger cell cycle arrest and apoptosis. The Bloom syndrome DNA helicase BLM interacts with the MMR complex, suggesting functional relevance to repair and checkpoint responses. We observed a strong interaction of BLM with MMR proteins in HeLa cells upon treatment with MNU as evidenced by co-immunoprecipitation as well as colocalization in the nucleus as revealed by dual immunofluorescence staining. Knockout of BLM sensitized HeLa MR cells to MNU-induced cell cycle disruption and enhanced expression of the apoptosis markers cleaved caspase-9 and PARP1. MNU-treated BLM-deficient cells also exhibited a greater number of 53BP1 foci and greater phosphorylation levels of H2AX at S139 and RPA32 at S8, indicating the accumulation of DNA double-strand breaks. These findings suggest that BLM prevents double-strand DNA breaks during the MMR-dependent DNA damage response and mitigates O6-meG-induced apoptosis.

RecQ helicase expression in patients with telomeropathies.

BACKGROUND: Telomeropathies are a group of inherited disorders caused by germline pathogenic variants in genes involved in telomere maintenance, resulting in excessive telomere attrition that affects several tissues, including hematopoiesis. RecQ and RTEL1 helicases contribute to telomere maintenance by unwinding telomeric structures such as G-quadruplexes (G4), preventing replication defects. Germline RTEL1 variants also are etiologic in telomeropathies. METHODS AND RESULTS: Here we investigated the expression of RecQ (RECQL1, BLM, WRN, RECQL4, and RECQL5) and RTEL1 helicase genes in peripheral blood mononuclear cells (PBMCs) from human telomeropathy patients. The mRNA expression levels of all RecQ helicases, but not RTEL1, were significantly downregulated in patients' primary cells. Reduced RecQ expression was not attributable to cell proliferative exhaustion, as RecQ helicases were not attenuated in T cells exhausted in vitro. An additional fifteen genes involved in DNA damage repair and RecQ functional partners also were downregulated in the telomeropathy cells. CONCLUSION: These findings indicate that the expression of RecQ helicases and functional partners involved in DNA repair is downregulated in PBMCs of telomeropathy patients.

Suppressors of Blm-deficiency identify three novel proteins that facilitate DNA repair in Ustilago maydis.

To identify new molecular components of the Brh2-governed homologous recombination (HR)-network in the highly radiation-resistant fungus Ustilago maydis, we undertook a genetic screen for suppressors of blm-KR hydroxyurea (HU)-sensitivity. Twenty DNA-damage sensitive mutants were obtained, three of which showing slow-growth phenotypes. Focusing on the "normally" growing candidates we identified five mutations, two in previously well-defined genes (Rec2 and Rad51) and the remaining three in completely uncharacterized genes (named Rec3, Bls9 and Zdr1). A common feature among these novel factors is their prominent role in DNA repair. Rec3 contains the P-loop NTPase domain which is most similar to that found in U. maydis Rec2 protein, and like Rec2, Rec3 plays critical roles in induced allelic recombination, is crucial for completion of meiosis, and with regard to DNA repair Δrec3 and Δrec2 are epistatic to one another. Importantly, overexpression of Brh2 in Δrec3 can effectively restore DNA-damage resistance, indicating a close functional connection between Brh2 and Rec3. The Bls9 does not seem to have any convincing domains that would give a clue as to its function. Nevertheless, we present evidence that, besides being involved in DNA-repair, Bls9 is also necessary for HR between chromosome homologs. Moreover, Δbls9 showed epistasis with Δbrh2 with respect to killing by DNA-damaging agents. Both, Rec3 and Bls9, play an important role in protecting the genome from mutations. Zdr1 is Cys2-His2 zinc finger (C2H2-ZF) protein, whose loss does not cause a detectable change in HR. Also, the functions of both Bls9 and Zdr1 genes are dispensable in meiosis and sporulation. However, Zdr1 appears to have overlapping activities with Blm and Mus81 in protecting the organism from methyl methanesulfonate- and diepoxybutane-induced DNA-damage. Finally, while deletion of Rec3 and Zdr1 can suppress HU-sensitivity of blm-KR, Δgen1, and Δmus81 mutants, interestingly loss of Bls9 does not rescue HU-sensitivity of Δgen1.

Characterization and regulation of cell cycle-independent noncanonical gene targeting.

Homology-dependent targeted DNA integration, generally referred to as gene targeting, provides a powerful tool for precise genome modification; however, its fundamental mechanisms remain poorly understood in human cells. Here we reveal a noncanonical gene targeting mechanism that does not rely on the homologous recombination (HR) protein Rad51. This mechanism is suppressed by Rad52 inhibition, suggesting the involvement of single-strand annealing (SSA). The SSA-mediated gene targeting becomes prominent when DSB repair by HR or end-joining pathways is defective and does not require isogenic DNA, permitting 5% sequence divergence. Intriguingly, loss of Msh2, loss of BLM, and induction of a target-site DNA break all significantly and synergistically enhance SSA-mediated targeted integration. Most notably, SSA-mediated integration is cell cycle-independent, occurring in the G1 phase as well. Our findings provide unequivocal evidence for Rad51-independent targeted integration and unveil multiple mechanisms to regulate SSA-mediated targeted as well as random integration.

Expression of human RECQL5 in Saccharomyces cerevisiae causes transcription defects and transcription-associated genome instability.

RECQL5 is a member of the conserved RecQ family of DNA helicases involved in the maintenance of genome stability that is specifically found in higher eukaryotes and associates with the elongating RNA polymerase II. To expand our understanding of its function we expressed human RECQL5 in the yeast Saccharomyces cerevisiae, which does not have a RECQL5 ortholog. We found that RECQL5 expression leads to cell growth inhibition, increased genotoxic sensitivity and transcription-associated hyperrecombination. Chromatin immunoprecipitation and transcriptomic analysis of yeast cells expressing human RECQL5 shows that this is recruited to transcribed genes and although it causes only a weak impact on gene expression, in particular at G + C-rich genes, it leads to a transcription termination defect detected as readthrough transcription. The data indicate that the interaction between RNAPII and RECQL5 is conserved from yeast to humans. Unexpectedly, however, the RECQL5-ID mutant, previously shown to have reduced the association with RNAPII in vitro, associates with the transcribing polymerase in cells. As a result, expression of RECQL5-ID leads to similar although weaker phenotypes than wild-type RECQL5 that could be transcription-mediated. Altogether, the data suggests that RECQL5 has the intrinsic ability to function in transcription-dependent and independent genome dynamics in S. cerevisiae.

RECQL4 is not critical for firing of human DNA replication origins.

Human RECQL4, a member of the RecQ helicase family, plays a role in maintaining genomic stability, but its precise function remains unclear. The N-terminus of RECQL4 has similarity to Sld2, a protein required for the firing of DNA replication origins in budding yeast. Consistent with this sequence similarity, the Xenopus laevis homolog of RECQL4 has been implicated in initiating DNA replication in egg extracts. To determine whether human RECQL4 is required for firing of DNA replication origins, we generated cells in which both RECQL4 alleles were targeted, resulting in either lack of protein expression (knock-out; KO) or expression of a full-length, mutant protein lacking helicase activity (helicase-dead; HD). Interestingly, both the RECQL4 KO and HD cells were viable and exhibited essentially identical origin firing profiles as the parental cells. Analysis of the rate of fork progression revealed increased rates in the RECQL4 KO cells, which might be indicative of decreased origin firing efficiency. Our results are consistent with human RECQL4 having a less critical role in firing of DNA replication origins, than its budding yeast homolog Sld2.

A Recql5 mutant facilitates complex CRISPR/Cas9-mediated chromosomal engineering in mouse zygotes.

Complex chromosomal rearrangements (CCRs) are often observed in clinical samples from patients with cancer and congenital diseases but are difficult to induce experimentally. Here, we report the first success in establishing animal models for CCRs. Mutation in Recql5, a crucial member of the DNA helicase RecQ family involved in DNA replication, transcription, and repair, enabled CRISPR/Cas9-mediated CCRs, establishing a mouse model containing triple fusion genes and megabase-sized inversions. Some of these structural features of individual chromosomal rearrangements use template switching and microhomology-mediated break-induced replication mechanisms and are reminiscent of the newly described phenomenon "chromoanasynthesis." These data show that Recql5 mutant mice could be a powerful tool to analyze the pathogenesis of CCRs (particularly chromoanasynthesis) whose underlying mechanisms are poorly understood. The Recql5 mutants generated in this study are to be deposited at key animal research facilities, thereby making them accessible for future research on CCRs.

BLM helicase unwinds lagging strand substrates to assemble the ALT telomere damage response.

The Bloom syndrome (BLM) helicase is critical for alternative lengthening of telomeres (ALT), a homology-directed repair (HDR)-mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. The DNA substrates that BLM engages to direct telomere recombination during ALT remain unknown. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT-positive cells to assemble a replication-associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2, commensurate with the appearance of telomere C-strand-specific single-stranded DNA (ssDNA). DNA2 nuclease deficiency increased 5'-flap formation in a BLM-dependent manner, while telomere C-strand, but not G-strand, nicks promoted ALT. These findings define the seminal events in the ALT DNA damage response, linking aberrant telomeric lagging strand DNA replication with a BLM-directed HDR mechanism that sustains telomere length in a subset of human cancers.

A case of Bloom syndrome manifesting with therapy-related myelodysplastic syndromes harboring a novel BLM gene variant.

Bloom syndrome (BS) is an autosomal recessive genetic disorder caused by variants in the BLM gene. BS is characterized by distinct facial features, elongated limbs, and various dermatological complications including photosensitivity, poikiloderma, and telangiectatic erythema. The BLM gene encodes a RecQ helicase critical for genome maintenance, stability, and repair, and a deficiency in functional BLM protein leads to genomic instability and high predisposition to various types of cancers, particularly hematological and gastrointestinal malignancies. Here, we report a case of BS with a previously unreported variant in the BLM gene. The patient was a 34-year-old woman who presented with short stature, prominent facial features, and a history of malignancies, including lymphoma, breast cancer, and myelodysplastic syndromes (MDS). She was initially treated with azacitidine for MDS and showed transient improvement, but eventually died at age of 35 due to progression of MDS. Genetic screening revealed compound heterozygous variants in the BLM gene, with a recurrent variant previously reported in BS in one allele and a previously unreported variant in the other allele. Based on her characteristic clinical features and the presence of heterozygous variants in the BLM gene, she was diagnosed with BS harboring compound heterozygous BLM variants.

RECQL4 Inhibits Radiation-Induced Tumor Immune Awakening via Suppressing the cGAS-STING Pathway in Hepatocellular Carcinoma.

Many patients with hepatocellular carcinoma (HCC) respond poorly to radiotherapy despite remarkable advances in treatment. A deeper insight into the mechanism of sensitivity of HCC to this therapy is urgently required. It is demonstrated that RECQL4 is upregulated in the malignant cells of patients with HCC. Elevated RECQL4 levels reduce the sensitivity of HCC to radiotherapy by repairing radiation-induced double-stranded DNA (dsDNA) fragments. Mechanistically, the inhibitory effect of RECQL4 on radiotherapy is due to the reduced recruitment of dendritic cells and CD8+ T cells in the tumor microenvironment (TME). RECQL4 disrupts the radiation-induced transformation of the TME into a tumoricidal niche by inhibiting the cGAS-STING pathway in dendritic cells. Knocking out STING in dendritic cells can block the impact of RECQL4 on HCC radiosensitivity. Notably, high RECQL4 expressions in HCC is significantly associated with poor prognosis in multiple independent cohorts. In conclusion, this study highlights how HCC-derived RECQL4 disrupts cGAS-STING pathway activation in dendritic cells through DNA repair, thus reducing the radiosensitivity of HCC. These findings provide new perspectives on the clinical treatment of HCC.

1,6-Hexanediol Is Inducing Homologous Recombination by Releasing BLM from Assemblysomes in Drosophila melanogaster.

We recently demonstrated that 1,6-hexanediol inhibits the formation of assemblysomes. These membraneless cell organelles have important roles in co-translational protein complex assembly and also store halfway translated DNA damage response proteins for a timely stress response. Recognizing the therapeutic potential of 1,6-hexanediol in dismantling assemblysomes likely to be involved in chemo- or radiotherapy resistance of tumor cells, we initiated an investigation into the properties of 1,6-hexanediol. Our particular interest was to determine if this compound induces DNA double-strand breaks by releasing the BLM helicase. Its yeast ortholog Sgs1 was confirmed to be a component of assemblysomes. The BLM helicase induces DNA damage when overexpressed due to the DNA double-strand breaks it generates during its normal function to repair DNA damage sites. It is evident that storing Sgs1 helicase in assemblysomes is crucial to express the full-length functional protein only in the event of DNA damage. Alternatively, if we dissolve assemblysomes using 1,6-hexanediol, ribosome-nascent chain complexes might become targets of ribosome quality control. We explored these possibilities and found, through the Drosophila wing-spot test assay, that 1,6-hexanediol induces DNA double-strand breaks. Lethality connected to recombination events following 1,6-hexanediol treatment can be mitigated by inducing DNA double-strand breaks with X-ray. Additionally, we confirmed that SMC5 recruits DmBLM to DNA damage sites, as knocking it down abolishes the rescue effect of DNA double-strand breaks on 1,6-hexanediol-induced lethality in Drosophila melanogaster.

TFIP11 promotes replication fork reversal to preserve genome stability.

Replication fork reversal, a critical protective mechanism against replication stress in higher eukaryotic cells, is orchestrated via a series of coordinated enzymatic reactions. The Bloom syndrome gene product, BLM, a member of the highly conserved RecQ helicase family, is implicated in this process, yet its precise regulation and role remain poorly understood. In this study, we demonstrate that the GCFC domain-containing protein TFIP11 forms a complex with the BLM helicase. TFIP11 exhibits a preference for binding to DNA substrates that mimic the structure generated at stalled replication forks. Loss of either TFIP11 or BLM leads to the accumulation of the other protein at stalled forks. This abnormal accumulation, in turn, impairs RAD51-mediated fork reversal and slowing, sensitizes cells to replication stress-inducing agents, and enhances chromosomal instability. These findings reveal a previously unidentified regulatory mechanism that modulates the activities of BLM and RAD51 at stalled forks, thereby impacting genome integrity.

BLM and BRCA1-BARD1 coordinate complementary mechanisms of joint DNA molecule resolution.

The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1, and RMI2 to form the BTR complex, which dissolves double Holliday junctions and DNA replication intermediates to promote sister chromatid disjunction before cell division. In its absence, structure-specific nucleases like the SMX complex (comprising SLX1-SLX4, MUS81-EME1, and XPF-ERCC1) can cleave joint DNA molecules instead, but cells deficient in both BTR and SMX are not viable. Here, we identify a negative genetic interaction between BLM loss and deficiency in the BRCA1-BARD1 tumor suppressor complex. We show that this is due to a previously overlooked role for BARD1 in recruiting SLX4 to resolve DNA intermediates left unprocessed by BLM in the preceding interphase. Consequently, cells with defective BLM and BRCA1-BARD1 accumulate catastrophic levels of chromosome breakage and micronucleation, leading to cell death. Thus, we reveal mechanistic insights into SLX4 recruitment to DNA lesions, with potential clinical implications for treating BRCA1-deficient tumors.

Massive contractions of myotonic dystrophy type 2-associated CCTG tetranucleotide repeats occur via double-strand break repair with distinct requirements for DNA helicases.

Myotonic dystrophy type 2 (DM2) is a genetic disease caused by expanded CCTG DNA repeats in the first intron of CNBP. The number of CCTG repeats in DM2 patients ranges from 75 to 11,000, yet little is known about the molecular mechanisms responsible for repeat expansions or contractions. We developed an experimental system in Saccharomyces cerevisiae that enables the selection of large-scale contractions of (CCTG)100 within the intron of a reporter gene and subsequent genetic analysis. Contractions exceeded 80 repeat units, causing the final repetitive tract to be well below the threshold for disease. We found that Rad51 and Rad52 are involved in these massive contractions, indicating a mechanism that uses homologous recombination. Srs2 helicase was shown previously to stabilize CTG, CAG, and CGG repeats. Loss of Srs2 did not significantly affect CCTG contraction rates in unperturbed conditions. In contrast, loss of the RecQ helicase Sgs1 resulted in a 6-fold decrease in contraction rate with specific evidence that helicase activity is required for large-scale contractions. Using a genetic assay to evaluate chromosome arm loss, we determined that CCTG and reverse complementary CAGG repeats elevate the rate of chromosomal fragility compared to a short-track control. Overall, our results demonstrate that the genetic control of CCTG repeat contractions is notably distinct among disease-causing microsatellite repeat sequences.

Werner helicase mediates the senescence and cell cycle of leukemia cells by regulating DNA repair pathways.

Leukemia is a type of malignant hematological disease that is generally resistant to chemotherapy and has poor therapeutic outcomes. Werner (WRN) DNA helicase, an important member of the RecQ family of helicases, plays an important role in DNA repair and telomere stability maintenance. WRN gene dysfunction leads to premature aging and predisposes humans to various types of cancers. However, the biological function of WRN in cancer remains unknown. In this study, the expression of this RecQ family helicase was investigated in different types of leukemia cells, and the leukemia cell line K562 with high WRN expression was selected to construct a WRN knockdown cell line. The results showed that WRN knockdown inhibited leukemia occurrence and development by regulating the proliferation, cell cycle, differentiation, and aging of cells and other biological processes. The results of transcriptome sequencing revealed that WRN promoted the sensitivity of leukemia cells to the DNA damage inducer Etoposide by regulating cell cycle-related proteins, such as CDC2, cyclin B1, p16, and p21, as well as key proteins in DNA damage repair pathways, such as p53, RAD50, RAD51, and MER11. Our findings show that WRN helicase is a promising potential target for leukemia treatment, providing new ideas for the development of targeted drugs against leukemia.

Immune infiltration, aggressive pathology, and poor survival outcomes in RECQL helicase deficient breast cancers.

RECQL is essential for genomic stability. Here, we evaluated RECQL in 449 pure ductal carcinomas in situ (DCIS), 152 DCIS components of mixed DCIS/invasive breast cancer (IBC) tumors, 157 IBC components of mixed DCIS/IBC and 50 normal epithelial terminal ductal lobular units (TDLUs). In 726 IBCs, CD8+, FOXP3+, IL17+, PDL1+, PD1+ T-cell infiltration (TILs) were investigated in RECQL deficient and proficient cancers. Tumor mutation burden (TMB) was evaluated in five RECQL germ-line mutation carriers with IBC by genome sequencing. Compared with normal epithelial cells, a striking reduction in nuclear RECQL in DCIS was evident with aggressive pathology and poor survival. In RECQL deficient IBCs, CD8+, FOXP3+, IL17+ or PDL1+ TILs were linked with aggressive pathology and shorter survival. In germline RECQL mutation carriers, increased TMB was observed in 4/5 tumors. We conclude that RECQL loss is an early event in breast cancer and promote immune cell infiltration.