RecQ Helicase Family Research Articles

Comprehensive in silico characterization of Arabidopsis thaliana RecQl helicases through structure prediction and molecular dynamics simulations

Helicases are ubiquitous enzymes with specific functions that contribute to almost all nucleic acid metabolic processes. The RecQ helicase family is essential for integrity in all organisms through DNA replication, repair, and recombination. This study investigated five RecQ-like helicases in Arabidopsis thaliana (AtRecQl) that exhibit diverse structural and physiochemical attributes and functions. Cis-regulatory element analysis identified stress, hormone, cell cycle, and development-responsive modules involved in various events in plant growth and development. Gene ontology analysis revealed that the five AtRecQl were associated with various cellular components, molecular functions, and biological processes. Protein-protein interaction analysis also implicated some in various abiotic stress processes. Structural analysis and molecular dynamics (MD) simulations were performed to examine conformational stability through root means square deviation and radius of gyration, showing stable AtRecQl protein structures. Free energy landscape analysis validated thermodynamically stable structures throughout the MD simulation. Principle component analysis and probability density functions from MD simulations provided satisfactory structural variational data for the complexes and limited coordinate movements. These insights might greatly benefit future studies.

Chromosomal breakage and sister chromatid exchange analysis in breast cancer patients with heterozygous BLM gene variants

Abstract Objectives BLM, a member of the RecQ helicase family, plays an important role in DNA repair, and its biallelic mutations cause autosomal recessive Bloom syndrome, a disease characterized by elevated levels of sister chromatid exchange (SCE) in affected individuals and hereditary cancer susceptibility in carriers. This study aims to investigate genomic instability in breast cancer patients carrying heterozygous variants in the BLM gene. Methods Spontaneous chromosome breakage count and SCE counting were performed on newly drawn blood cultures, both spontaneous and stimulated. The spontaneous breakage count was conducted alongside control samples. In SCE analysis, 0–10 per metaphase was considered normal, 10–40 borderline, and counts above 40 were considered high. Results The study included 26 patients and one healthy control at each session. The clinical and pathological characteristics of the patients were evaluated. The analyses revealed borderline-level increased SCE rates in only one patient. No increase in spontaneous breakage count or SCE analysis was observed in other individuals compared to controls. Conclusions Increased genomic instability was not observed in the analyzed patient group. These results can lead to multiple interpretations. The variants carried in the BLM gene in the patient group may be of low pathogenicity, or increased instability compared to controls may not be necessary for heterozygous variants. Additionally, our patient group may not have been exposed to a genotoxic effect causing genomic instability. These results could also indicate a favorable position in terms of avoiding chemotherapy and radiotherapy complications.

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.

Abstract 7602: Discovery of GH1581, a potent and selective WRN inhibitor as a cancer therapy

Abstract Defects in DNA mismatch repair (MMR) proteins could induce damage of genome, resulting microsatellite instability (MSI). MSI is highly correlated with the occurrence of several types of cancers, mainly in colon, gastric, endometrial, and ovarian cancers. Despite advances in the treatment of MSI-high cancers, tumor evolution and drug resistance remain the leading causes of treatment failure and death in cancer patients. About 50% MSI cancers have no response to immune checkpoint inhibitors. There is still unmet needs to be fulfilled for MSI-high patients. WRN is a RecQ family helicase involves in genome stability maintenance, DNA repair, replication, transcription, and telomere maintenance. Recent studies have identified WRN as a synthetic lethal target of MSI-high cancers. Employing structure-based drug design strategy, we have discovered a potent and selective WRN inhibitor, GH1581. In vitro, GH1581 displayed specific < 10 nM potency in ADP-Glo assay and < 10 nM potency in cell proliferation assay using MSI cancer cell lines. In vivo, GH1581 demonstrated favorable pharmacokinetic properties in different species. In SW48 xenograft model, it inhibited the tumor growth with 100% TGI at QD dosing of 10 mg/kg without severe adverse effect on body weight. In summary, we discovered GH1581 as a novel inhibitor of WRN with high in vitro and in vivo efficacy. Citation Format: Jiapeng Li, Guiping Zhang, Jie Jack Li. Discovery of GH1581, a potent and selective WRN inhibitor as a cancer therapy [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 7602.

Abstract 5921: PH027-1, a potent and selective small-molecule WRN inhibitor that targets MSI-H tumors

Abstract Background: High microsatellite instability (MSI-H) is highly prevalent across many cancer types including endometrium, gastric, ovary and colorectal cancer. It is well known that there is a clear synthetic lethality (SL) interaction between MSI-H and Werner syndrome protein (WRN) inhibition. WRN is a member of RecQ helicase family and involved in the maintenance of genome integrity. Different from other RecQ family members, WRN has two activity centers responsible for helicase and exonuclease activity, respectively. Further studies confirmed that an inhibition on helicase activity, but not exonuclease activity, is responsible for SL interaction with MSI-H. Also, Recent clinical observations supported that though loss-of-function of WRN could cause an early-aged disease Werner syndrome, selective loss of helicase but not exonuclease leads to only mild to moderate symptoms, suggesting a selective inhibitor of helicase activity has the potential to induce the SL interaction with MSI-H and keep patients safe. Methods: Biochemical assays and cell-based assays were performed to evaluate in vitro activity and selectivity of PH027-1. The in vivo anti-tumor efficacy was tested in two cell-derived xenograft (CDX) mouse models with tumors harboring MSI-H. The in vivo pharmacokinetics (PK) properties were evaluated in mice, rats and dogs, and other in vitro and in vivo PK and safety properties were assessed with corresponding assay methods. Results: Biochemical assays showed that PH027-1 potently inhibited helicase activity of WRN (IC50, 0.8 nM), but had no effect on other RecQ family members even at the highest tested concentration of 10 μM (IC50, all≥10 μM for BLM, RecQ4 and RecQ5). At the same time, PH027-1 did not affect the exonuclease activity of WRN at all tested concentrations. In cell-based assays, PH027-1 potently inhibited cell viability of MSI-H cancer cells SW48 and HCT116 at tens of nM, but had no effect on MSS cells (HT29 cancer cells and CD34+ hematopoietic stem cells) even at 10 μM. The tests were further expanded to 18 cancer cell lines covering uterus, gastric, ovary and colorectal cancer. Nine of twelve MSI-H cell lines were very sensitive to WRN inhibition, and all six MSS cell lines were totally resistant to the treatment. PH027-1 possessed excellent PK profiles as shown by in vivo PK studies, with absolute bioavailability of 58%~102% in mice, rats and dogs. In two CDX models (SW48 and HCT116), oral administration of PH027-1 dose-dependently inhibited tumor growth, and a nearly complete inhibition was observed at a dose of 20 mg/kg QD. Tumor regression was clearly shown at higher doses. All other in vitro and in vivo PK and safety tests showed favorable results and supported for preclinical development of PH027-1. Conclusion: As a highly selective and potent inhibitor on helicase activity of WRN, PH027-1 has the potential to become a novel and safe approach for treatment of MSI-H tumors. Citation Format: Feng Gao, Bin Liu, Liandong Jing, Jing Wang, Yongyong Wu, Jun Wan, Pengzhi Zhang, Yu Gao, Zhizhong Li, Yongqi Guo. PH027-1, a potent and selective small-molecule WRN inhibitor that targets MSI-H tumors [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 5921.

Abstract 7278: Discovery of a novel WRN inhibitor, ZM-3329 that efficiently inhibits MSI-H tumor growth

Abstract Microsatellite instability-high status (MSI-H) is typically associated with endometrial and gastrointestinal cancers. These tumors harbor large numbers of frameshift and single-nucleotide variants and are characterized by a high tumor mutational burden (TMB). The clinical benefits of immune checkpoint inhibitor (ICI) therapy on MSI-H cancers were well-demonstrated, however, 50% of these cancers are intrinsically resistant to PD-1 therapies and resistance inevitably develops with prolonged treatment. Thus, a novel efficient therapeutic strategy for MSI-H cancer is still needed. Werner syndrome ATP-dependent helicase (WRN) is a member of the RecQ helicases family and has recently emerged as a promising synthetic-lethal target in the treatment of MSI-H tumors. In MSI-H tumors, microsatellite sequences consisting of repeated nucleotides are unstable, and replication slippage causes a unique secondary structure that hinders the progression of replication forks. With its helicase activity, WRN unwinds this secondary structure to restart DNA synthesis and sustain MSI-H tumor cell growth. WRN knockdown or helicase enzyme-dead effectively inhibits cell proliferation and tumor growth in multiple preclinical MSI-H models. Here we identified ZM-3329, a novel and highly potent WRN inhibitor. Helicase activities were efficiently inhibited by ZM-3329 with IC50 less than 30 nM, and equal potency was identified on HCT-116 cell proliferation inhibition. In a panel of 124 tumor cell lines, ZM-3329 only specifically inhibited the growth of MSI-H but had no significant effects on the MSS (Microsatellite stable) tumor cell lines, indicating high selectivity against MSS. Additionally, ZM-3329 strongly inhibited the growth of MSI-H patient-derived organoids. The intracellular mechanism of action study revealed that inhibition of WRN by ZM-3329 in MSI-H cells led to the accumulative DNA damage signatures which was demonstrated by the increase of γH2AX and P21 expression, and arrested cell cycle to G2/M. ZM-3329 demonstrated favorable pharmacokinetic profiles in pre-clinical species, supporting oral administration in humans. ZM-3329 showed robust anti-tumor activities in multiple MSI-H xenograft models with significant tumor regression observed. Meanwhile, mice were tolerant well in all tested doses, and no abnormality was observed. In conclusion, a novel WRN inhibitor was developed, and robust antitumor activities were demonstrated in xenograft tumors and PDOs with different tissue types. In terms of synthetic lethality between MSI-H and WRN as well as broad anti-tumor activities across different tumor types, ZM-3329 would be developed as a tissue-agnostic therapy for MSI-H patients. Citation Format: Feng Zhou, Guimei Yang, Yajing Liu, Liting Xue, Yao Guo, Zhengtao Li, Weikun Wang, Jian Li, Renhong Tang. Discovery of a novel WRN inhibitor, ZM-3329 that efficiently inhibits MSI-H tumor growth [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 7278.

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.

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.

A Novel Quinazoline Derivative Prevents and Treats Arsenic-Induced Liver Injury by Regulating the Expression of RecQ Family Helicase.

Arsenic is a carcinogenic metalloid toxicant widely found in the natural environment. Acute or prolonged exposure to arsenic causes a series of damages to the organs, mainly the liver, such as hepatomegaly, liver fibrosis, cirrhosis, and even hepatocellular carcinoma. Therefore, it is imperative to seek drugs to prevent arsenic-induced liver injury. Quinazolines are a class of nitrogen heterocyclic compounds with biological and pharmacological effects in vivo and in vitro. This study was designed to investigate the ameliorating effects of quinazoline derivatives on arsenic-induced liver injury and its molecular mechanism. We investigated the mechanism of the quinazoline derivative KZL-047 in preventing and ameliorating arsenic-induced liver injury in vitro by cell cycle and apoptosis. We performed real-time fluorescence quantitative polymerase chain reaction (qPCR) and Western blotting combined with molecular docking. In vivo, the experiments were performed to investigate the mechanism of KZL-047 in preventing and ameliorating arsenic-induced liver injury using arsenic-infected mice. Physiological and biochemical indices of liver function in mouse serum were measured, histopathological changes in liver tissue were observed, and immunohistochemical staining was used to detect changes in the expression of RecQ-family helicases in mouse liver tissue. The results of in vitro experiments showed that sodium arsenite (SA) inhibited the proliferation of L-02 cells, induced apoptosis, blocked the cell cycle at the G1 phase, and decreased the expression of RecQ family helicase; after KZL-047 treatment in arsenic-induced L-02 cells, the expression of RecQ family helicase was upregulated, and the apoptosis rate was slowed, leading to the restoration of the cell viability level. KZL-047 inhibited arsenic-induced oxidative stress, alleviated oxidative damage and lipid peroxidation in vivo, and ameliorated arsenic toxicity-induced liver injury. KZL-047 restored the expression of RecQ family helicase proteins, which is consistent with the results of in vitro studies. In summary, KZL-047 can be considered a potential candidate for the treatment of arsenic-induced liver injury.

Understanding the Human RECQ5 Helicase-Connecting the Dots from DNA to Clinics.

RECQ5, a member of the conserved RECQ helicase family, is the sole human RECQ homolog that has not been linked to a hereditary developmental syndrome. Nonetheless, dysregulation of RECQ5 has emerged as a significant clinical concern, being linked to cancer predisposition, cardiovascular disease, and inflammation. In cells, RECQ5 assumes a crucial role in the regulation of DNA repair pathways, particularly in the repair of DNA double-strand breaks and inter-strand DNA crosslinks. Moreover, RECQ5 exhibits a capacity to modulate gene expression by interacting with transcription machineries and their co-regulatory proteins, thus safeguarding against transcription-induced DNA damage. This review aims to provide an overview of the multifaceted functions of RECQ5 and its implications in maintaining genomic stability. We will discuss the potential effects of clinical variants of RECQ5 on its cellular functions and their underlying mechanisms in the pathogenesis of cancer and cardiovascular disease. We will review the impact of RECQ5 variants in the field of pharmacogenomics, specifically their influence on drug responses, which may pave the way for novel therapeutic interventions targeting RECQ5 in human diseases.

Biochemical properties of naturally occurring human bloom helicase variants.

Bloom syndrome helicase (BLM) is a RecQ-family helicase implicated in a variety of cellular processes, including DNA replication, DNA repair, and telomere maintenance. Mutations in human BLM cause Bloom syndrome (BS), an autosomal recessive disorder that leads to myriad negative health impacts including a predisposition to cancer. BS-causing mutations in BLM often negatively impact BLM ATPase and helicase activity. While BLM mutations that cause BS have been well characterized both in vitro and in vivo, there are other less studied BLM mutations that exist in the human population that do not lead to BS. Two of these non-BS mutations, encoding BLM P868L and BLM G1120R, when homozygous, increase sister chromatid exchanges in human cells. To characterize these naturally occurring BLM mutant proteins in vitro, we purified the BLM catalytic core (BLMcore, residues 636-1298) with either the P868L or G1120R substitution. We also purified a BLMcore K869A K870A mutant protein, which alters a lysine-rich loop proximal to the P868 residue. We found that BLMcore P868L and G1120R proteins were both able to hydrolyze ATP, bind diverse DNA substrates, and unwind G-quadruplex and duplex DNA structures. Molecular dynamics simulations suggest that the P868L substitution weakens the DNA interaction with the winged-helix domain of BLM and alters the orientation of one lobe of the ATPase domain. Because BLMcore P868L and G1120R retain helicase function in vitro, it is likely that the increased genome instability is caused by specific impacts of the mutant proteins in vivo. Interestingly, we found that BLMcore K869A K870A has diminished ATPase activity, weakened binding to duplex DNA structures, and less robust helicase activity compared to wild-type BLMcore. Thus, the lysine-rich loop may have an important role in ATPase activity and specific binding and DNA unwinding functions in BLM.

Abstract 1628: A small-molecule inhibitor of WRN selectively kills MSI-H cancer cells and phenocopies WRN genetic defects

Abstract Werner syndrome protein (WRN) is a RecQ-family helicase involved in the maintenance of genome integrity. Germline mutations in WRN cause premature aging and cancer predisposition. Analysis of systematic RNAi and CRISPR screening data has previously revealed that WRN is essential for the survival of cancer cell lines with high microsatellite instability (MSI-H). We have developed potent and selective small-molecule inhibitors of WRN helicase (WRNi) and showed that pharmacological inhibition of WRN causes lethality and induction of DNA damage markers selectively in MSI-H cancer cell lines compared to microsatellite-stable (MSS) cell lines. Screening of WRNi across a large panel of pooled, barcoded cell lines in the PRISM format revealed selective sensitivity in MSI-H cell lines and showed that pharmacological inhibition of WRN is highly correlated with genetic ablation of WRN across this panel, confirming selectivity for WRN. In vivo evaluation demonstrated robust and MSI-selective tumor regressions. These data provide pharmacological proof-of-concept for the WRN/MSI-H synthetic lethal relationship and support WRN inhibition as a novel therapeutic approach for the treatment of MSI-H cancers. Citation Format: Yanhua Rao, Anjana Srivatsan, Marya Liimatta, Diana Munoz, Jeanne Quirit, Jianxia Shi, An Nguyen, Xin Linghu, Federowicz Federowicz, Brian Jones, Melissa Fleury, Zach Newby, Michael P. Dillon, Paul A. Barsanti, Mark R. Lackner, Michael A. White, Yang Lee, Phil Landis, Yang Peng, Michelle Cicchini, Josh Cottom, Leng Nickels, Ed Brnardic, Michael DeMartino, Josh Taygerly. A small-molecule inhibitor of WRN selectively kills MSI-H cancer cells and phenocopies WRN genetic defects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1628.

RecQ dysfunction contributes to social and depressive-like behavior and affects aldolase activity in mice

RecQ helicase family proteins play vital roles in maintaining genome stability, including DNA replication, recombination, and DNA repair. In human cells, there are five RecQ helicases: RECQL1, Bloom syndrome (BLM), Werner syndrome (WRN), RECQL4, and RECQL5. Dysfunction or absence of RecQ proteins is associated with genetic disorders, tumorigenesis, premature aging, and neurodegeneration. The biochemical and biological roles of RecQ helicases are rather well established, however, there is no systematic study comparing the behavioral changes among various RecQ-deficient mice including consequences of exposure to DNA damage. Here, we investigated the effects of ionizing irradiation (IR) on three RecQ-deficient mouse models (RecQ1, WRN and RecQ4). We find abnormal cognitive behavior in RecQ-deficient mice in the absence of IR. Interestingly, RecQ dysfunction impairs social ability and induces depressive-like behavior in mice after a single exposure to IR, suggesting that RecQ proteins play roles in mood and cognition behavior. Further, transcriptomic and metabolomic analyses revealed significant alterations in RecQ-deficient mice, especially after IR exposure. In particular, pathways related to neuronal and microglial functions, DNA damage repair, cell cycle, and reactive oxygen responses were downregulated in the RecQ4 and WRN mice. In addition, increased DNA damage responses were found in RecQ-deficient mice. Notably, two genes, Aldolase Fructose-Bisphosphate B (Aldob) and NADPH Oxidase 4 (Nox4), were differentially expressed in RecQ-deficient mice. Our findings suggest that RecQ dysfunction contributes to social and depressive-like behaviors in mice, and that aldolase activity may be associated with these changes, representing a potential therapeutic target.

A non-catalytic N-terminus domain of WRN prevents mitotic telomere deprotection

Telomeric ends form a loop structure (T-loop) necessary for the repression of ATM kinase activation throughout the normal cell cycle. However, cells undergoing a prolonged mitotic arrest are prone to lose the T-loop, resulting in Aurora B kinase-dependent mitotic telomere deprotection, which was proposed as an anti-tumor mechanism that eliminates precancerous cells from the population. The mechanism of mitotic telomere deprotection has not been elucidated. Here, we show that WRN, a RECQ helicase family member, can suppress mitotic telomere deprotection independently of its exonuclease and helicase activities. Truncation of WRN revealed that N-terminus amino acids 168–333, a region that contains a coiled-coil motif, is sufficient to suppress mitotic telomere deprotection without affecting both mitotic Aurora B-dependent spindle checkpoint and ATM kinase activity. The suppressive activity of the WRN168–333 fragment is diminished in cells partially depleted of TRF2, while WRN is required for complete suppression of mitotic telomere deprotection by TRF2 overexpression. Finally, we found that phosphomimetic but not alanine mutations of putative Aurora B target sites in the WRN168–333 fragment abolished its suppressive effect. Our findings reveal a non-enzymatic function of WRN, which may be regulated by phosphorylation in cells undergoing mitotic arrest. We propose that WRN enhances the protective function of TRF2 to counteract the hypothetical pathway that resolves the mitotic T-loop.

Werner helicase is required for proliferation and DNA damage repair in multiple myeloma

Multiple myeloma (MM), characterized by extensive genomic instability and aberrant DNA damage repair, is a plasma cell malignancy due to the excessive proliferation of monoclonal antibody-producing plasma cells in the bone marrow. Despite the significant improvement in the survival of patients with the development of novel therapeutic agents, MM remains an incurable disease. Werner (WRN) helicase, a member of the RecQ helicase family that contributes to DNA replication, recombination, and repair, has been highlighted in cancer cell survival, yet the role and mechanism of WRN in MM remain unclear. Increased mRNA expression of WRN in newly diagnosed and relapsed CD138+ myeloma plasma cells than normal CD138+ plasma cells and their matched CD138- non-tumorigenic cells were detected by qPCR. Using NSC19630, a specific WRN helicase inhibitor, we further showed decreased cell viability, proliferation, and DNA repair and increased DNA damage and apoptosis in MM cells by MTT assay, cell cycle assay, apoptosis assay, and Western blotting. The results of the present study demonstrate that WRN is essential in MM cell viability, proliferation, and genomic stability, indicating its inhibition may enhance the efficacy of chemotherapy in MM.

Bloom Syndrome Patients Display Accelerated Epigenetic Aging in Blood Lymphocytes

Bloom Syndrome (BSyn) is an autosomal recessive disorder caused by variants in the BLM genes at locus 15q26.1. The BLM gene encodes a protein that is part of the DNA RECQ helicase family, which has a role in homologous recombination, DNA structural integrity, DNA repair responses, and telomere maintenance. Patients with BSyn present with poor growth, sun sensitivity, mild immunodeficiency, diabetes, and increased risk of cancer, most commonly leukemias. Additionally, patients with BSyn develop myelodysplasia and life-threatening side effects from standard chemotherapy and radiation, often due to bone marrow toxicity. Cells from patients with BSyn display increased sister chromatid exchange, telomere defects, and telomere-dependent chromosome fusions. Despite these aging-associated findings, patients with BSyn do not appear to have typical premature aging characteristics such as graying hair, osteoporosis, and early-onset cardiovascular disease. We hypothesized that patients with BSyn may have increased epigenetic aging in blood, which may be related to their increased risk of hematologic dysfunction and malignancies. In this study, we aimed to use DNA methylation in order to identify an aging signature that could be a potential biomarker of disease risk. In partnership with the Bloom Syndrome Registry (BSR) as well as JScreen and our own clinical practice, we extracted 49 DNA samples from blood lymphocytes of patients with BSyn as well as carriers. The samples were then processed per standard Illumina protocol for the Infinium MethylationEPIC BeadChip kit. We utilized the online DNA Methylation Age Calculator (https://dnamage.genetics.ucla.edu/) to calculate multiple measures of methylation aging for each patient, including the Horvath pan-tissue epigenetic clock DNAmAge, the Hannum clock, the GrimAge lifespan predictor, and the PhenoAge phenotypic age estimate. Simple linear regression was used to determine statistical significance. Of the 49 total samples, 18 samples were from patients with BSyn (ages 1-38 years) and 31 samples were from carriers with one variant in BLM (ages 23-69 years). We discovered that BSyn patients had increased DNAm age compared to carriers measured by the pan-tissue clock (p=0.0054). Similarly, epigenetic clocks that were designed to predict human mortality risk (GrimAge and PhenoAge) indicate significant age acceleration as well, p<0.0088 and p<0.0001 respectively. Finally, DNA methylation-based estimators of telomere length show significantly shorter telomere lengths in BSyn patients compared to carriers, p<0.0001. Epigenetic age can be a better predictor of health and disease over biologic or chronologic age. In this study, we show for the first time that patients with BSyn have evidence of epigenetic aging across several measures in blood lymphocytes. Future studies are ongoing to investigate underlying mechanisms for this and whether epigenetic aging in these patients can predict hematologic dysfunction and cancer risk.

A Large Case-Control Study Performed in Spanish Population Suggests That RECQL5 Is the Only RECQ Helicase Involved in Breast Cancer Susceptibility.

Simple SummaryAround 50% of the familial breast cancer (BC) cases are estimated to be caused by variants in low-, moderate-, and high-risk susceptibility genes; however, the other half is of unknown origin. The finding of new susceptibility genes is key to improve diagnosis, take preventive measures, and identify new therapies. In this context, previous studies have discussed whether the genes encoding for the RECQ helicase family could play a role in BC susceptibility, without very conclusive results. To clarify this, in this study, we sequenced the whole coding sequence of the RECQL1, BLM, WRN, RECQL4, and RECQL5 genes in 1993 Spanish BC familial cases and compared it with controls from gnomAD. No association was found for RECQL1, BLM, WRN, and RECQL4; however, we did find an association between RECQL5 and breast cancer as a moderate-risk gene, making it a perfect candidate for further studies.Around 50% of the familial breast cancer (BC) cases are estimated to be caused by germline variants in known low-, moderate-, and high-risk susceptibility genes, while the other half is of unknown genetic origin. In the present study, we wanted to evaluate the role of the RECQ helicases, some of which have been studied in the past as candidates, with unclear results about their role in the disease. Using next-generation sequencing (NGS) technology, we analyzed the whole coding sequence of BLM, RECQL1, RECQL4, RECQL5, and WRN in almost 2000 index cases from BC Spanish families that had previously tested negative for the known BC susceptibility genes (BRCAX) and compared the results with the controls extracted from gnomAD. Our results suggest that BLM, RECQL1, RECQL4, and WRN do not play a major role in BC susceptibility. However, in the combined analysis, joining the present results with those previously reported in a series of 1334 BC Spanish patients and controls, we found a statistically significant association between Loss of Function (LoF) variants in RECQL5 and BC risk, with an OR of 2.56 (p = 0.009; 95% CI, 1.18–4.98). Our findings support our previous work and places the RECQL5 gene as a new moderate-risk BC gene.

Bloom Syndrome Helicase Compresses Single‐Stranded DNA into Phase‐Separated Condensates

Bloom syndrome protein (BLM) is a conserved RecQ family helicase involved in the maintenance of genome stability. BLM has been widely recognized as a genome “caretaker” that processes structured DNA. In contrast, our knowledge of how BLM behaves on single-stranded (ss) DNA is still limited. Here, we demonstrate that BLM possesses the intrinsic ability for phase separation and can co-phase separate with ssDNA to form dynamically arrested protein/ssDNA co-condensates. The introduction of ATP potentiates the capability of BLM to condense on ssDNA, which further promotes the compression of ssDNA against a resistive force of up to 60 piconewtons. Moreover, BLM is also capable of condensing replication protein A (RPA)- or RAD51-coated ssDNA, before which it generates naked ssDNA by dismantling these ssDNA-binding proteins. Overall, our findings identify an unexpected characteristic of a DNA helicase and provide a new angle of protein/ssDNA co-condensation for understanding the genomic instability caused by BLM overexpression under diseased conditions.

Bloom Syndrome Helicase Compresses Single-Stranded DNA into Phase-Separated Condensates.

Bloom syndrome protein (BLM) is a conserved RecQ family helicase involved in the maintenance of genome stability. BLM has been widely recognized as a genome "caretaker" that processes structured DNA. In contrast, our knowledge of how BLM behaves on single-stranded (ss) DNA is still limited. Here, we demonstrate that BLM possesses the intrinsic ability for phase separation and can co-phase separate with ssDNA to form dynamically arrested protein/ssDNA co-condensates. The introduction of ATP potentiates the capability of BLM to condense on ssDNA, which further promotes the compression of ssDNA against a resistive force of up to 60 piconewtons. Moreover, BLM is also capable of condensing replication protein A (RPA)- or RAD51-coated ssDNA, before which it generates naked ssDNA by dismantling these ssDNA-binding proteins. Overall, our findings identify an unexpected characteristic of a DNA helicase and provide a new angle of protein/ssDNA co-condensation for understanding the genomic instability caused by BLM overexpression under diseased conditions.

Genetic and biochemical interactions of yeast DNA helicases.

DNA helicases function in many types of nucleic acid transactions, and as such, they are vital for genome integrity. Although they are often considered individually, work from many groups demonstrates that these enzymes often genetically and biochemically interact in vivo. Here, we highlight methods to interrogate such interactions among the PIF1 (Pif1 and Rrm3) and RecQ (Hrq1 and Sgs1) family helicases in Saccharomyces cerevisiae. The interactions among these enzymes were investigated in vivo using deletion and inactivation alleles with a gross-chromosomal rearrangement (GCR) assay. Further, wild-type and inactive recombinant proteins were used to determine the effects of the helicases on telomerase activity in vitro. We found that synergistic increases in GCR rates often occur in double vs. single mutants, suggesting that the helicases function in distinct genome integrity pathways. Further, the recombinant helicases can function together in vitro to modulate telomerase activity. Overall, the data suggest that the interactions among the members of these DNA helicase families are multipartite and argue for a comprehensive systems biology approach to fully elucidate the physiological interplay between these enzymes.