Explore the vast range of titles available.

G-quadruplex (GQ) is a four stranded DNA secondary structure that arises from a guanine rich sequence. Stable formation of GQ in genomic DNA can be counteracted by the resolving activity of specialized helicases including RNA helicase AU (associated with AU rich elements) (RHAU) (G4 resolvase 1), Bloom helicase (BLM), and Werner helicase (WRN). However, their substrate specificity and the mechanism involved in GQ unfolding remain uncertain. Here, we report that RHAU, BLM, and WRN exhibit distinct GQ conformation specificity, but use a common mechanism of repetitive unfolding that leads to disrupting GQ structure multiple times in succession. Such unfolding activity of RHAU leads to efficient annealing exclusively within the same DNA molecule. The same resolving activity is sufficient to dislodge a stably bound GQ ligand, including BRACO-19, NMM, and Phen-DC3. Our study demonstrates a plausible biological scheme where different helicases are delegated to resolve specific GQ structures by using a common repetitive unfolding mechanism that provides a robust resolving power.

RecQ4 is a member of the RecQ helicase family, an evolutionarily conserved class of enzymes, dedicated to preserving genomic integrity by operating in telomere maintenance, DNA repair and replication. While reduced RecQ4 activity is associated with cancer predisposition and premature aging, RecQ4 upregulation is related to carcinogenesis and metastasis. Within the RecQ family, RecQ4 assumes an exceptional position, lacking several characteristic RecQ domains. Here we present the crystal structure of human RecQ4, encompassing the conserved ATPase core and a novel C-terminal domain that lacks resemblance to the RQC domain observed in other RecQ helicases. The new domain features a zinc-binding site and two distinct types of winged-helix domains, which are not involved in canonical DNA binding or helicase activity. Based on our structural and functional analysis, we propose that RecQ4 exerts a helicase mechanism, which may be more closely related to bacterial RecQ helicases than to its human family members. RecQ helicases are important for maintaining genomic integrity. Here, the authors present functional data and the crystal structure of human RecQ4, which exerts a helicase mechanism that may be more closely related to bacterial RecQ helicases than to its human family members.

Owing to their crucial role in genome maintenance, RecQ helicases are ubiquitous and present across organisms. Though the multiplicity of RecQ helicases is well known in higher organisms, it is rare among bacteria. The ancient cyanobacterium Nostoc sp. strain PCC7120 was found to have three annotated RecQ helicases. This study aims at understanding its structural differences and evolution through bioinformatics approach and functionality through expression analysis studies. Nostoc RecQ helicases were found to be transcriptionally regulated by LexA and DNA damage inducing stresses. Bioinformatic analysis revealed that all three RecQ helicases of Nostoc possess helicases_C and Zn +2 -binding domains. Two of the helicases (AnRecQ and AnRecQ2) lacked the complete RQC and HRDC domains, and AnRecQ2 had an additional Phosphoribosyl transferase domain (Pribosyltran), also seen in RecQ-like helicase (RqlH) protein of Mycobacterium smegmatis . AnRecQ1, which was similar to most bacterial RecQ helicases, differed in having a long C-terminal tail. STRING analysis revealed that the proteins also differed in their predicted protein interactome. Phylogenetic analysis suggested that the multiple recQ genes may have been acquired through duplication and acquisition of additional domains from the smallest of the RecQ helicases (AnRecQ) to cater multiple functions required to deal with the harsh environmental conditions. In course of evolution, however, the multiplicity was lost with the modern-day bacteria and lower eukaryotes which retained fewer RecQ helicases, while further duplication of the acquired RECQ occurred in higher animals and plants to deal with cellular complexity.

Homeostatic regulation of G-quadruplexes (G4s), four-stranded structures that can form in guanine-rich nucleic acids, requires G4 unwinding helicases. The mechanisms that mediate G4 unwinding remain unknown. We report the structure of a bacterial RecQ DNA helicase bound to resolved G4 DNA. Unexpectedly, a guanine base from the unwound G4 is sequestered within a guanine-specific binding pocket. Disruption of the pocket in RecQ blocks G4 unwinding, but not G4 binding or duplex DNA unwinding, indicating its essential role in structure-specific G4 resolution. A novel guanine-flipping and sequestration model that may be applicable to other G4-resolving helicases emerges from these studies. How G-quadruplexes (G4s) are resolved by helicases is still a matter of investigation. Here the authors provide mechanistic insight into G4s unwinding by presenting a crystal structure of resolved G4 DNA and the G4 binding domain of RecQ helicase from the bacterium Cronobacter sakazakii .

Cells must continuously repair inevitable DNA damage while avoiding the deleterious consequences of imprecise repair. Distinction between legitimate and illegitimate repair processes is thought to be achieved in part through differential recognition and processing of specific noncanonical DNA structures, although the mechanistic basis of discrimination remains poorly defined. Here, we show that Escherichia coli RecQ, a central DNA recombination and repair enzyme, exhibits differential processing of DNA substrates based on their geometry and structure. Through single-molecule and ensemble biophysical experiments, we elucidate how the conserved domain architecture of RecQ supports geometry-dependent shuttling and directed processing of recombination-intermediate [displacement loop (D-loop)] substrates. Our study shows that these activities together suppress illegitimate recombination in vivo, whereas unregulated duplex unwinding is detrimental for recombination precision. Based on these results, we propose a mechanism through which RecQ helicases achieve recombination precision and efficiency.

Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive disorder characterized by skin rash (poikiloderma), skeletal dysplasia, small stature, juvenile cataracts, sparse or absent hair, and predisposition to specific malignancies such as osteosarcoma and hematological neoplasms. RTS is caused by germ-line mutations in RECQL4, a RecQ helicase family member. In vitro studies have identified functions for the ATP-dependent helicase of RECQL4. However, its specific role in vivo remains unclear. To determine the physiological requirement and the biological functions of Recql4 helicase activity, we generated mice with an ATP-binding-deficient knock-in mutation (Recql4K525A). Recql4K525A/K525A mice were strikingly normal in terms of embryonic development, body weight, hematopoiesis, B and T cell development, and physiological DNA damage repair. However, mice bearing two distinct truncating mutations Recql4G522Efs and Recql4R347*, that abolished not only the helicase but also the C-terminal domain, developed a profound bone marrow failure and decrease in survival similar to a Recql4 null allele. These results demonstrate that the ATP-dependent helicase activity of Recql4 is not essential for its physiological functions and that other domains might contribute to this phenotype. Future studies need to be performed to elucidate the complex interactions of RECQL4 domains and its contribution to the development of RTS.

G-quadruplex (G4) structures are highly stable four-stranded DNA and RNA secondary structures held together by non-canonical guanine base pairs. G4 sequence motifs are enriched at specific sites in eukaryotic genomes, suggesting regulatory functions of G4 structures during different biological processes. Considering the high thermodynamic stability of G4 structures, various proteins are necessary for G4 structure formation and unwinding. In a yeast one-hybrid screen, we identified Slx9 as a novel G4-binding protein. We confirmed that Slx9 binds to G4 DNA structures in vitro. Despite these findings, Slx9 binds only insignificantly to G-rich/G4 regions in Saccharomyces cerevisiae as demonstrated by genome-wide ChIP-seq analysis. However, Slx9 binding to G4s is significantly increased in the absence of Sgs1, a RecQ helicase that regulates G4 structures. Different genetic and molecular analyses allowed us to propose a model in which Slx9 recognizes and protects stabilized G4 structures in vivo.

Significance RecQ DNA helicases catalyze critical genome maintenance reactions in nearly all organisms. This study describes the crystal structure of a bacterial RecQ helicase bound in a productive complex with DNA. Together with biochemical experiments, the structure reveals a conserved coupling mechanism that links DNA binding to ATP hydrolysis in RecQ enzymes. These findings also help explain how structural dynamics could facilitate RecQ’s noted ability to process diverse DNA substrates. A model explaining the physical basis for RecQ substrate binding and unwinding is proposed. RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. Here, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3′ single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots ∼90° to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATP hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. This bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures.

The Ku-binding motif (KBM) is a short peptide module first identified in APLF that we now show is also present in Werner syndrome protein (WRN) and in Modulator of retrovirus infection homologue (MRI). We also identify a related but functionally distinct motif in XLF, WRN, MRI and PAXX, which we denote the XLF-like motif. We show that WRN possesses two KBMs; one at the N terminus next to the exonuclease domain and one at the C terminus next to an XLF-like motif. We reveal that the WRN C-terminal KBM and XLF-like motif function cooperatively to bind Ku complexes and that the N-terminal KBM mediates Ku-dependent stimulation of WRN exonuclease activity. We also show that WRN accelerates DSB repair by a mechanism requiring both KBMs, demonstrating the importance of WRN interaction with Ku. These data define a conserved family of KBMs that function as molecular tethers to recruit and/or stimulate enzymes during NHEJ. Werner syndrome is a progeroid disease characterised by genetic instability due to mutations to the WRN helicase/exonuclease. Here the authors define a novel Ku binding motif (KBM) and show that two such motifs facilitate the involvement of WRN in DNA double-strand break repair.

The BLM helicase protein plays a vital role in DNA replication and the maintenance of genomic integrity. Variation in the BLM helicase gene resulted in defects in the DNA repair mechanism and was reported to be associated with Bloom syndrome (BS) and cancer. Despite extensive investigation of helicase proteins in humans, no attempt has previously been made to comprehensively analyse the single nucleotide polymorphism (SNPs) of the BLM gene. In this study, a comprehensive analysis of SNPs on the BLM gene was performed to identify, characterize and validate the pathogenic SNPs using computational approaches. We obtained SNP data from the dbSNP database version 150 and mapped these data to the genomic coordinates of the “NM_000057.3” transcript expressing BLM helicase (P54132). There were 607 SNPs mapped to missense, 29 SNPs mapped to nonsense, and 19 SNPs mapped to 3′-UTR regions. Initially, we used many consensus tools of SIFT, PROVEAN, Condel, and PolyPhen-2, which together increased the accuracy of prediction and identified 18 highly pathogenic non-synonymous SNPs (nsSNPs) out of 607 SNPs. Subsequently, these 18 high-confidence pathogenic nsSNPs were analysed for BLM protein stability, structure–function relationships and disease associations using various bioinformatics tools. These 18 mutants of the BLM protein along with the native protein were further investigated using molecular dynamics simulations to examine the structural consequences of the mutations, which might reveal their malfunction and contribution to disease. In addition, 28 SNPs were predicted as “stop gained” nonsense SNPs and one SNP was predicted as “start lost”. Two SNPs in the 3′UTR were found to abolish miRNA binding and thus may enhance the expression of BLM. Interestingly, we found that BLM mRNA overexpression is associated with different types of cancers. Further investigation showed that the dysregulation of BLM is associated with poor overall survival (OS) for lung and gastric cancer patients and hence led to the conclusion that BLM has the potential to be used as an important prognostic marker for the detection of lung and gastric cancer.