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.

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.

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.

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.

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.

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.

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.

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.

Bisbenzylisoquinoline alkaloid fangchinoline derivative HY-2 inhibits breast cancer cells by suppressing BLM DNA helicase.

The high expression of BLM (Bloom syndrome) DNA helicase in tumors involves its strong association with cell expansion. Bisbenzylisoquinoline alkaloids own an antitumor property and have developed as candidates for anticancer drugs. This paper aimed to study the antitumor effect of fangchinoline derivative HY-2 by targeting BLM642-1290 DNA helicase, and then explore its inhibitory mechanism on proliferation of MDA-MB-435 breast cancer cells. We confirmed that the mRNA and protein levels of BLM DNA helicase in breast cancer were higher than those in normal tissues. HY-2 could inhibit the DNA binding, ATPase and DNA unwinding of BLM642-1290 DNA helicase with enzymatic assay. HY-2 could also inhibit the DNA unwinding of DNA helicase in cells. In addition, HY-2 showed an inhibiting the MDA-MB-435, MDA-MB-231, MDA-MB-436 breast cancer cells expansion. The mRNA and protein levels of BLM DNA helicase in MDA-MB-435 cells increased after HY-2 treatment, which might contribute to HY-2 inhibiting the DNA binding, ATPase and DNA unwinding of BLM DNA helicase. The mechanism of HY-2 inhibition on BLM DNA helicase was further confirmed with the effect of HY-2 on the ultraviolet spectrogram of BLM642-1290 DNA helicase and Molecular dynamics simulation of the interacting between HY-2 and BLM640-1291 DNA helicase. Our study provided some valuable clues for the exploration of HY-2 in the living body and developing it as an anticancer drug.

Comprehensive mapping of cell fates in microsatellite unstable cancer cells supports dual targeting of WRN and ATR.

Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN-knowledge that would be helpful for informing clinical development of WRN targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system in which the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We found that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we found no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low-dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provide the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggest that dual targeting of WRN and ATR might be a useful strategy for treating MSI-H cancers.

Modified iPOND revealed the role of mutant p53 in promoting helicase function and telomere maintenance.

The G-rich DNA, such as telomere, tends to form G-quadruplex (G4) structure, which slows down the replication fork progression, induces replication stress, and becomes the chromosome fragile sites. Here we described a molecular strategy that cells developed to overcome the DNA replication stress via DNA helicase regulation. The p53N236S (p53S) mutation has been found in the Werner syndrome mouse embryo fibroblast (MEFs) escaped from senescence, could be the driving force for cell escaping senescence. We revealed that the p53S could transcriptionally up-regulate DNA helicases expression, including Wrn, Blm, Timeless, Ddx, Mcm, Gins, Fanc, as well as telomere specific proteins Terf1, Pot1, through which p53S promoted the unwinding of G4 structures, and protected the cells from DNA replication stress induced by G4 stabilizer. By modified iPOND (isolation of proteins on nascent DNA) assay and telomere assay, we demonstrated that the p53S could promote the recruitment of those helicases to the DNA replication forks, facilitated the maintenance of telomere, and prevent the telomere dysfunction induced by G4 stabilizer. Interestingly, we did not observe the function of promoting G4 resolving and facilitating telomere lengthening in the cells with Li-Fraumeni Syndrome mutation-p53R172H (p53H), which suggests that this is the specific gain of function for p53S. Together our data suggest that the p53S could gain the new function of releasing the replication stress via regulating the helicase function and G4 structure, which benefits telomere lengthening. This strategy could be applied to the treatment of diseases caused by telomere replication stress.

Genomic Instability of G-Quadruplex Sequences in Escherichia coli: Roles of DinG, RecG, and RecQ Helicases.

Guanine-rich DNA can fold into highly stable four-stranded DNA structures called G-quadruplexes (G4). Originally identified in sequences from telomeres and oncogene promoters, they can alter DNA metabolism. Indeed, G4-forming sequences represent obstacles for the DNA polymerase, with important consequences for cell life as they may lead to genomic instability. To understand their role in bacterial genomic instability, different G-quadruplex-forming repeats were cloned into an Escherichia coli genetic system that reports frameshifts and complete or partial deletions of the repeat when the G-tract comprises either the leading or lagging template strand during replication. These repeats formed stable G-quadruplexes in single-stranded DNA but not naturally supercoiled double-stranded DNA. Nevertheless, transcription promoted G-quadruplex formation in the resulting R-loop for (G3T)4 and (G3T)8 repeats. Depending on genetic background and sequence propensity for structure formation, mutation rates varied by five orders of magnitude. Furthermore, while in vitro approaches have shown that bacterial helicases can resolve G4, it is still unclear whether G4 unwinding is important in vivo. Here, we show that a mutation in recG decreased mutation rates, while deficiencies in the structure-specific helicases DinG and RecQ increased mutation rates. These results suggest that G-quadruplex formation promotes genetic instability in bacteria and that helicases play an important role in controlling this process in vivo.

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.

Mannich Base PIP-199 Is a Chemically Unstable Pan-Assay Interference Compound.

Mannich base PIP-199 is the only reported small-molecule inhibitor of the Fanconi anemia complementation group M-RecQ-mediated genome instability protein (FANCM-RMI), a protein-protein interaction that governs genome instability in the genetic disorders Fanconi anemia and Bloom's syndrome. PIP-199 and analogues with the same indole-derived Mannich base scaffold have been used as tool compounds in diverse biological studies. We report the first published synthesis of PIP-199 and its analogues, demonstrating that PIP-199 immediately decomposes in common aqueous buffers and some organic solvents. Neither PIP-199 nor its more hydrolytically stable analogues show any observable activity in binding and competitive biophysical assays for FANCM-RMI. We conclude that PIP-199 is not an effective tool compound for biological studies and that apparent cellular activity likely arises from the nonspecific toxicity of breakdown products. More generally, apparent inhibitors that share this Mannich scaffold potentially represent a new family of pan-assay interference compounds (PAINS) that should be thoroughly assessed for aqueous stability prior to use in biological studies.

Functional inhibition of RECQL5 helicase elicits non-homologous end joining response and sensitivity of breast cancers to PARP inhibitor.

Poly (ADPRibose) Polymerase inhibitor (PARPi) are clinically approved for the treatment of BRCA-mutated hereditary breast and ovarian cancers with homologous recombination (HR) deficiency, based on synthetic lethality concept. However, ∼90% of breast cancers are BRCA-wild type; they repair PARPi mediated damage through HR, leading to intrinsic de novo resistance. Hence, there is an unmet need of exploring novel targets in HR-proficient aggressive breast cancers for PARPi treatment. RECQL5 physically interacts and disrupts RAD51 from pre-synaptic filaments, aiding HR resolution, replication fork protection and preventing illegitimate recombination. In the current investigation, we show that targeted inhibition of HR by stabilization of RAD51-RECQL5 complex by a pharmacological inhibitor of RECQL5 (4a; 1,3,4-oxadiazole derivative) in the presence of PARPi [talazoparib (BMN673)] leads to abolition of functional HR with uncontrolled activation of NHEJ repair. This was assessed by GFP based NHEJ reporter assay, KU80 recruitment and in vitro NHEJ based plasmid ligation assay. Concomitant treatment with talazoparib and 4a generates copious amounts of replication stress, prolonged cell cycle arrest, extensive double strand breaks (DSBs) and mitotic catastrophe, leading to sensitization of HR-proficient breast cancers. Suppression of NHEJ activity abolishes 4a-mediated sensitization of breast cancers to PARPi treatment. Imperatively, 4a was ineffective against normal mammary epithelial cells, which expresses low RECQL5 vis-à-vis breast cancer cells. Moreover, functional inhibition of RECQL5 suppresses metastatic potential of breast cancer cells in response to PARPi. Together, we identified RECQL5 as a novel pharmacological target for expanding PARPi based treatment horizon for HR-proficient cancers.

Phase-separated ribosome-nascent chain complexes in genotoxic stress response.

Assemblysomes are EDTA- and RNase-resistant ribonucleoprotein (RNP) complexes of paused ribosomes with protruding nascent polypeptide chains. They have been described in yeast and human cells for the proteasome subunit Rpt1, and the disordered amino-terminal part of the nascent chain was found to be indispensable for the accumulation of the Rpt1-RNP into assemblysomes. Motivated by this, to find other assemblysome-associated RNPs we used bioinformatics to rank subunits of Saccharomyces cerevisiae protein complexes according to their amino-terminal disorder propensity. The results revealed that gene products involved in DNA repair are enriched among the top candidates. The Sgs1 DNA helicase was chosen for experimental validation. We found that indeed nascent chains of Sgs1 form EDTA-resistant RNP condensates, assemblysomes by definition. Moreover, upon exposure to UV, SGS1 mRNA shifted from assemblysomes to polysomes, suggesting that external stimuli are regulators of assemblysome dynamics. We extended our studies to human cell lines. The BLM helicase, ortholog of yeast Sgs1, was identified upon sequencing assemblysome-associated RNAs from the MCF7 human breast cancer cell line, and mRNAs encoding DNA repair proteins were overall enriched. Using the radiation-resistant A549 cell line, we observed by transmission electron microscopy that 1,6-hexanediol, an agent known to disrupt phase-separated condensates, depletes ring ribosome structures compatible with assemblysomes from the cytoplasm of cells and makes the cells more sensitive to X-ray treatment. Taken together, these findings suggest that assemblysomes may be a component of the DNA damage response from yeast to human.

A Novel Germline Pathogenic Variant of RECQL4 Gene in an Iranian Pedigree with Familial Squamous Cell Carcinoma: A Brief Report.

Squamous cell carcinoma (SCC) is the most common human solid tumor and the leading cause of cancer death. SCC of the breast is a very rare type of cancer that has not been well researched. Early identification of the genetic factors involved can lead to early diagnosis and targeted treatment. The present study was conducted in 2018 at Isfahan University of Medical Sciences (Isfahan, Iran). The proband was a 66-year-old woman with SCC of the breast and a positive family history of cancer. Blood DNA samples were used for whole-exome sequencing to identify germline pathogenic variants. Variant annotation and prioritization were done on variant call format files using bioinformatics software tools. The screened variants were confirmed using the Sanger sequencing method. Co-segregation analysis was performed on the blood DNA samples of the first- and second-degree relatives of the proband to assess the presence of the mutation. A novel germline pathogenic variant was identified in the RECQL4 gene of the family. RECQL4 is a known protein in DNA repair and replication. Considering its effect on other types of SCC, it may play an important role in SCC initiation and progression in the breast.

Hyperubiquitylation of DNA helicase RECQL4 by E3 ligase MITOL prevents mitochondrial entry and potentiates mitophagy.

Mutations in the DNA helicase RECQL4 lead to Rothmund-Thomson syndrome (RTS), a disorder characterized by mitochondrial dysfunctions, premature aging, and genomic instability. However, the mechanisms by which these mutations lead to pathology are unclear. Here we report that RECQL4 is ubiquitylated by a mitochondrial E3 ligase, MITOL, at two lysine residues (K1101, K1154) via K6 linkage. This ubiquitylation hampers the interaction of RECQL4 with mitochondrial importer Tom20, thereby restricting its own entry into mitochondria. We show the RECQL4 2K mutant (where both K1101 and K1154 are mutated) has increased entry into mitochondria and demonstrates enhanced mitochondrial DNA (mtDNA) replication. We observed that the three tested RTS patient mutants were unable to enter the mitochondria and showed decreased mtDNA replication. Furthermore, we found that RECQL4 in RTS patient mutants are hyperubiquitylated by MITOL and form insoluble aggregate-like structures on the outer mitochondrial surface. However, depletion of MITOL allows RECQL4 expressed in these RTS mutants to enter mitochondria and rescue mtDNA replication. Finally, we show increased accumulation of hyperubiquitylated RECQL4 outside the mitochondria leads to the cells being potentiated to increased mitophagy. Hence, we conclude regulating the turnover of RECQL4 by MITOL may have a therapeutic effect in patients with RTS.

Identification of 2-Sulfonyl/Sulfonamide Pyrimidines as Covalent Inhibitors of WRN Using a Multiplexed High-Throughput Screening Assay.

Werner syndrome protein (WRN) is a multifunctional enzyme with helicase, ATPase, and exonuclease activities that are necessary for numerous DNA-related transactions in the human cell. Recent studies identified WRN as a synthetic lethal target in cancers characterized by genomic microsatellite instability resulting from defects in DNA mismatch repair pathways. WRN's helicase activity is essential for the viability of these high microsatellite instability (MSI-H) cancers and thus presents a therapeutic opportunity. To this end, we developed a multiplexed high-throughput screening assay that monitors exonuclease, ATPase, and helicase activities of full-length WRN. This screening campaign led to the discovery of 2-sulfonyl/sulfonamide pyrimidine derivatives as novel covalent inhibitors of WRN helicase activity. The compounds are specific for WRN versus other human RecQ family members and show competitive behavior with ATP. Examination of these novel chemical probes established the sulfonamide NH group as a key driver of compound potency. One of the leading compounds, H3B-960, showed consistent activities in a range of assays (IC50 = 22 nM, KD = 40 nM, KI = 32 nM), and the most potent compound identified, H3B-968, has inhibitory activity IC50 ∼ 10 nM. These kinetic properties trend toward other known covalent druglike molecules. Our work provides a new avenue for screening WRN for inhibitors that may be adaptable to different therapeutic modalities such as targeted protein degradation, as well as a proof of concept for the inhibition of WRN helicase activity by covalent molecules.

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.