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Several decades of research have identified Mcm10 hanging around the replisome making several critical contacts with a number of proteins but with no real disclosed function. Recently, the O'Donnell laboratory has been better able to map the interactions of Mcm10 with a larger Cdc45/GINS/MCM (CMG) unwinding complex placing it at the front of the replication fork. They have shown biochemically that Mcm10 has the impressive ability to strip off single-strand binding protein (RPA) and reanneal complementary DNA strands. This has major implications in controlling DNA unwinding speed as well as responding to various situations where fork reversal is needed. This work opens up a number of additional facets discussed here revolving around accessing the DNA junction for different molecular purposes within a crowded replisome. Abbreviations: alt-NHEJ: Alternative Nonhomologous End-Joining; CC: Coli-Coil motif; CMG: Cdc45/GINS/MCM2-7; CMGM: Cdc45/GINS/Mcm2-7/Mcm10; CPT: Camptothecin; CSB: Cockayne Syndrome Group B protein; CTD: C-Terminal Domain; DSB: Double-Strand Break; DSBR: Double-Strand Break Repair; dsDNA: Double-Stranded DNA; GINS: go-ichi-ni-san, Sld5-Psf1-Psf2-Psf3; HJ Dis: Holliday Junction dissolution; HJ Res: Holliday Junction resolution; HR: Homologous Recombination; ICL: Interstrand Cross-Link; ID: Internal Domain; MCM: Minichromosomal Maintenance; ND: Not Determined; NTD: N-Terminal Domain; PCNA: Proliferating Cell Nuclear Antigen; RPA: Replication Protein A; SA: Strand Annealing; SE: Strand Exchange; SEW: Steric Exclusion and Wrapping; ssDNA: Single-Stranded DNA; TCR: Transcription-Coupled Repair; TOP1: Topoisomerase [ABSTRACT FROM AUTHOR]

Timely recruitment of DNA damage response proteins to sites of genomic structural lesions is very important for signaling mechanisms to activate appropriate cell cycle checkpoints but also repair the altered DNA sequence to suppress mutagenesis. The eukaryotic cell is characterized by a complex cadre of players and pathways to ensure genomic stability in the face of replication stress or outright genomic insult by endogenous metabolites or environmental agents. Among the key performers are molecular motor DNA unwinding enzymes known as helicases that sense genomic perturbations and separate structured DNA strands so that replacement of a damaged base or sugar-phosphate backbone lesion can occur efficiently. Mutations in the BLM gene encoding the DNA helicase BLM leads to a rare chromosomal instability disorder known as Bloom’s syndrome. In a recent paper by the Sengupta lab, BLM’s role in the correction of double-strand breaks (DSB), a particularly dangerous form of DNA damage, was investigated. Adding to the complexity, BLM appears to be a key ringmaster of DSB repair as it acts both positively and negatively to regulate correction pathways of high or low fidelity. The FANCJ DNA helicase, mutated in another chromosomal instability disorder known as Fanconi Anemia, is an important player that likely coordinates with BLM in the balancing act. Further studies to dissect the roles of DNA helicases like FANCJ and BLM in DSB repair are warranted. [ABSTRACT FROM AUTHOR]

Cells are under constant assault from reactive oxygen species that occur endogenously or arise from environmental agents. An important consequence of such stress is the generation of oxidatively damaged DNA, which is represented by a wide range of non-helix distorting and helix-distorting bulkier lesions that potentially affect a number of pathways including replication and transcription; consequently DNA damage tolerance and repair pathways are elicited to help cells cope with the lesions. The cellular consequences and metabolism of oxidatively damaged DNA can be quite complex with a number of DNA metabolic proteins and pathways involved. Many of the responses to oxidative stress involve a specialized class of enzymes known as helicases, the topic of this review. Helicases are molecular motors that convert the energy of nucleoside triphosphate hydrolysis to unwinding of structured polynucleic acids. Helicases by their very nature play fundamentally important roles in DNA metabolism and are implicated in processes that suppress chromosomal instability, genetic disease, cancer, and aging. We will discuss the roles of helicases in response to nuclear and mitochondrial oxidative stress and how this important class of enzymes help cells cope with oxidatively generated DNA damage through their functions in the replication stress response, DNA repair, and transcriptional regulation. [ABSTRACT FROM AUTHOR]

Helicases and translocases use the energy of nucleoside triphosphate binding and hydrolysis to unwind/resolve structured nucleic acids or move along a single-stranded or double-stranded polynucleotide chain, respectively. These molecular motors facilitate a variety of transactions including replication, DNA repair, recombination, and transcription. A key partner of eukaryotic DNA helicases/translocases is the single-stranded DNA binding protein Replication Protein A (RPA). Biochemical, genetic, and cell biological assays have demonstrated that RPA interacts with these human molecular motors physically and functionally, and their association is enriched in cells undergoing replication stress. The roles of DNA helicases/translocases are orchestrated with RPA in pathways of nucleic acid metabolism. RPA stimulates helicase-catalyzed DNA unwinding, enlists translocases to sites of action, and modulates their activities in DNA repair, fork remodeling, checkpoint activation, and telomere maintenance. The dynamic interplay between DNA helicases/translocases and RPA is just beginning to be understood at the molecular and cellular levels, and there is still much to be learned, which may inform potential therapeutic strategies. [ABSTRACT FROM AUTHOR]

In 2010, a new recessive cohesinopathy disorder, designated Warsaw breakage syndrome (WABS), was described. The individual with WABS displayed microcephaly, pre- and postnatal growth retardation, and abnormal skin pigmentation. Cytogenetic analysis revealed mitomycin C (MMC)-induced chromosomal breakage; however, an additional sister chromatid cohesion defect was also observed. WABS is genetically linked to bi-allelic mutations in the ChlR1/ DDX11 gene which encodes a protein of the conserved family of Iron-Sulfur (Fe-S) cluster DNA helicases. Mutations in the budding yeast ortholog of ChlR1, known as Chl1, were known to cause sister chromatid cohesion defects, indicating a conserved function of the gene. In 2012, three affected siblings were identified with similar symptoms to the original WABS case, and found to have a homozygous mutation in the conserved Fe-S domain of ChlR1, confirming the genetic linkage. Significantly, the clinically relevant mutations perturbed ChlR1 DNA unwinding activity. In addition to its genetic importance in human disease, ChlR1 is implicated in papillomavirus genome maintenance and cancer. Although its precise functions in genome homeostasis are still not well understood, ongoing molecular studies of ChlR1 suggest the helicase plays a critically important role in cellular replication and/or DNA repair. [ABSTRACT FROM AUTHOR]

Abstract: Helicases have important roles in nucleic acid metabolism, and their prominence is marked by the discovery of genetic disorders arising from disease-causing mutations. Missense mutations can yield unique insight to molecular functions and basis for disease pathology. XPB or XPD missense mutations lead to Xeroderma pigmentosum, Cockayne''s syndrome, Trichothiodystrophy, or COFS syndrome, suggesting that DNA repair and transcription defects are responsible for clinical heterogeneity. Complex phenotypes are also observed for RECQL4 helicase mutations responsible for Rothmund–Thomson syndrome, Baller–Gerold syndrome, or RAPADILINO. Bloom''s syndrome causing missense mutations are found in the conserved helicase and RecQ C-terminal domain of BLM that interfere with helicase function. Although rare, patient-derived missense mutations in the exonuclease or helicase domain of Werner syndrome protein exist. Characterization of WRN separation-of-function mutants may provide insight to catalytic requirements for suppression of phenotypes associated with the premature aging disorder. Characterized FANCJ missense mutations associated with breast cancer or Fanconi anemia interfere with FANCJ helicase activity required for DNA repair and the replication stress response. For example, a FA patient-derived mutation in the FANCJ Iron-Sulfur domain was shown to uncouple its ATPase and translocase activity from DNA unwinding. Mutations in DDX11 (ChlR1) are responsible for Warsaw Breakage syndrome, a recently discovered autosomal recessive cohesinopathy. Ongoing and future studies will address clinically relevant helicase mutations and polymorphisms, including those that interfere with key protein interactions or exert dominant negative phenotypes (e.g., certain mutant alleles of Twinkle mitochondrial DNA helicase). Chemical rescue may be an approach to restore helicase activity in loss-of-function helicase disorders. Genetic and biochemical analyses of disease-causing missense mutations in human helicase disorders have led to new insights to the molecular defects underlying aberrant cellular and clinical phenotypes. [Copyright &y& Elsevier]

Alternate DNA structures that deviate from B-form double-stranded DNA such as G-quadruplex (G4) DNA can be formed by sequences that are widely distributed throughout the human genome. G-quadruplex secondary structures, formed by the stacking of planar quartets composed of four guanines that interact by Hoogsteen hydrogen bonding, can affect cellular DNA replication and transcription, and influence genomic stability. The unique metabolism of G-rich chromosomal regions that potentially form quadruplexes may influence a number of biological processes including immunoglobulin gene rearrangements, promoter activation and telomere maintenance. A number of human diseases are characterized by telomere defects, and it is proposed that G-quadruplex structures which form at telomere ends play an important role in telomere stability. Evidence from cellular studies and model organisms suggests that diseases with known defects in G4 DNA helicases are likely to be perturbed in telomere maintenance and cellular DNA replication. In this minireview, we discuss the connections of G-quadruplex nucleic acids to human genetic diseases and cancer based on the recent literature. [ABSTRACT FROM AUTHOR]

Abstract: Five human RecQ helicases (WRN, BLM, RECQ4, RECQ5, RECQ1) exist in humans. Of these, three are genetically linked to diseases of premature aging and/or cancer. Neither RECQ1 nor RECQ5 has yet been implicated in a human disease. However, cellular studies and genetic analyses of model organisms indicate that RECQ1 (and RECQ5) play an important role in the maintenance of genomic stability. Biochemical studies of purified RECQ1 protein demonstrate that the enzyme catalyzes DNA unwinding and strand annealing, and these activities are likely to be important for its role in DNA repair. RECQ1 also physically and functionally interacts with proteins involved in genetic recombination. In this review, we will summarize our current knowledge of RECQ1 roles in cellular nucleic acid metabolism and propose avenues of investigation for future studies. [Copyright &y& Elsevier]

Since the discovery of the DNA double helix, there has been a fascination in understanding the molecular mechanisms and cellular processes that account for: (i) the transmission of genetic information from one generation to the next and (ii) the remarkable stability of the genome. Nucleic acid biologists have endeavored to unravel the mysteries of DNA not only to understand the processes of DNA replication, repair, recombination, and transcription but to also characterize the underlying basis of genetic diseases characterized by chromosomal instability. Perhaps unexpectedly at first, DNA helicases have arisen as a key class of enzymes to study in this latter capacity. From the first discovery of ATP-dependent DNA unwinding enzymes in the mid 1970's to the burgeoning of helicase-dependent pathways found to be prevalent in all kingdoms of life, the story of scientific discovery in helicase research is rich and informative. Over four decades after their discovery, we take this opportunity to provide a history of DNA helicases. No doubt, many chapters are left to be written. Nonetheless, at this juncture we are privileged to share our perspective on the DNA helicase field – where it has been, its current state, and where it is headed. [ABSTRACT FROM AUTHOR]

Fanconi anemia (FA) is a hereditary chromosomal instability disorder often displaying congenital abnormalities and characterized by a predisposition to progressive bone marrow failure (BMF) and cancer. Over the last 25 years since the discovery of the first linkage of genetic mutations to FA, its molecular genetic landscape has expanded tremendously as it became apparent that FA is a disease characterized by a defect in a specific DNA repair pathway responsible for the correction of covalent cross-links between the two complementary strands of the DNA double helix. This pathway has become increasingly complex, with the discovery of now over 20 FA-linked genes implicated in interstrand cross-link (ICL) repair. Moreover, gene products known to be involved in double-strand break (DSB) repair, mismatch repair (MMR), and nucleotide excision repair (NER) play roles in the ICL response and repair of associated DNA damage. While ICL repair is predominantly coupled with DNA replication, it also can occur in non-replicating cells. DNA damage accumulation and hematopoietic stem cell failure are thought to contribute to the increased inflammation and oxidative stress prevalent in FA. Adding to its confounding nature, certain FA gene products are also engaged in the response to replication stress, caused endogenously or by agents other than ICL-inducing drugs. In this review, we discuss the mechanistic aspects of the FA pathway and the molecular defects leading to elevated replication stress believed to underlie the cellular phenotypes and clinical features of FA. [ABSTRACT FROM AUTHOR]

DNA helicases are molecular motors that harness the energy of nucleoside triphosphate hydrolysis to unwinding structured DNA molecules that must be resolved during cellular replication, DNA repair, recombination, and transcription. In vivo, DNA helicases are expected to encounter a wide spectrum of covalent DNA modifications to the sugar phosphate backbone or the nitrogenous bases; these modifications can be induced by endogenous biochemical processes or exposure to environmental agents. The frequency of lesion abundance can vary depending on the lesion type. Certain adducts such as oxidative base modifications can be quite numerous, and their effects can be helix-distorting or subtle perturbations to DNA structure. Helicase encounters with specific DNA lesions and more novel forms of DNA damage will be discussed. We will also review the battery of assays that have been used to characterize helicase-catalyzed unwinding of damaged DNA substrates. Characterization of the effects of specific DNA adducts on unwinding by various DNA repair and replication helicases has proven to be insightful for understanding mechanistic and biological aspects of helicase function in cellular DNA metabolism. [ABSTRACT FROM AUTHOR]

• Experimental approaches to assess primer extension assay using a G4 DNA template are described. • Experimental design of primer extension assays enables one to measure the extent and fidelity of DNA synthesis when a G4 obstacle is encountered. • Coordination of a G4-resolving helicase with a DNA polymerase can be assayed biochemically. • Biochemical studies to assess G4 DNA synthesis bypass aided by a G4-resolving helicase provides molecular insight that is relevant to genome metabolism and translational studies. G-quadruplex (G4) DNA poses a unique obstacle to DNA synthesis during replication or DNA repair due to its unusual structure which deviates significantly from the conventional DNA double helix. A mechanism to overcome the G4 roadblock is provided by the action of a G4-resolving helicase that collaborates with the DNA polymerase to smoothly catalyze polynucleotide synthesis past the unwound G4. In this technique-focused paper, we describe the experimental approaches of the primer extension assay using a G4 DNA template to measure the extent and fidelity of DNA synthesis by a DNA polymerase acting in concert with a G4-resolving DNA helicase. Important parameters pertaining to reaction conditions and controls are discussed to aid in the design of experiments and interpretation of the data obtained. This methodology can be applied in multiple capacities that may depend on the DNA substrate, DNA polymerase, or DNA helicase under investigation. [ABSTRACT FROM AUTHOR]

DNA helicases, known for their fundamentally important roles in genomic stability, are high profile players in cancer. Not only are there monogenic helicase disorders with a strong disposition to cancer, it is well appreciated that helicase variants are associated with specific cancers (e.g. , breast cancer). Flipping the coin, DNA helicases are frequently overexpressed in cancerous tissues and reduction in helicase gene expression results in reduced proliferation and growth capacity, as well as DNA damage induction and apoptosis of cancer cells. The seminal roles of helicases in the DNA damage and replication stress responses, as well as DNA repair pathways, validate their vital importance in cancer biology and suggest their potential values as targets in anti-cancer therapy. In recent years, many laboratories have characterized the specialized roles of helicase to resolve transcription-replication conflicts, maintain telomeres, mediate cell cycle checkpoints, remodel stalled replication forks, and regulate transcription. In vivo models, particularly mice, have been used to interrogate helicase function and serve as a bridge for preclinical studies that may lead to novel therapeutic approaches. In this review, we will summarize our current knowledge of DNA helicases and their roles in cancer, emphasizing the latest developments. [ABSTRACT FROM AUTHOR]

Characterizing the molecular deficiencies underlying human aging has been a formidable challenge as it is clear that a complex myriad of factors including genetic mutations, environmental influences, and lifestyle choices influence the deterioration responsible for human pathologies. In addition, the common denominators of human aging, exemplified by the newly updated hallmarks of aging (López-Otín et al., 2023), suggest multiple avenues and layers of crosstalk between pathways important for genome and cellular homeostasis, both of which are major determinants of both good health and lifespan. In this regard, we postulate that hereditary disorders characterized by chromosomal instability offer a unique window of insight into aging and age-related disease processes. Recently, we discovered a new RECQ helicase disorder, designated RECON syndrome attributed to bi-allelic mutations in the RECQL1 gene (Abu-Libdeh et al., 2022). Cells deficient in RECQL1 exhibit genomic instability and a compromised response to replication stress, providing further evidence for the significance of genome homeostasis to suppress disease phenotypes. Here we provide a perspective on the pathology of RECON syndrome to inform the reader as to how molecular defects in the RECQL1 gene contribute to underlying deficiencies in nucleic acid metabolism often seen in certain aging or age-related diseases. [Display omitted] • Hereditary premature aging disorders offer insight into aging and age-related disease. • RECON syndrome is attributed to bi-allelic mutations in the RECQL1 helicase gene. • Genomic instability and compromised replication are prevalent in RECON syndrome. • DNA damage underlies accelerated aging and by inference normal aging. [ABSTRACT FROM AUTHOR]

Abstract: The WRN gene defective in the premature aging disorder Werner syndrome encodes a helicase/exonuclease. We examined the ability of WRN to rescue DNA damage sensitivity of a yeast mutant defective in the Rad50 subunit of Mre11-Rad50-Xrs2 nuclease complex implicated in homologous recombination repair. Genetic studies revealed WRN operates in a yEXO1-dependent pathway to rescue rad50 sensitivity to methylmethane sulfonate (MMS). WRN helicase, but not exonuclease, is required for MMS resistance. WRN missense mutations in helicase or RecQ C-terminal domains interfered with the ability of WRN to rescue rad50 MMS sensitivity. WRN does not rescue rad50 ionizing radiation (IR) sensitivity, suggesting that WRN, in collaboration with yEXO1, is tailored to relieve replicational stress imposed by alkylated base damage. WRN and yEXO1 are associated with each other in vivo. Purified WRN stimulates hEXO1 nuclease activity on DNA substrates associated with a stalled or regressed replication fork. We propose WRN helicase operates in an EXO1-dependent pathway to help cells survive replicational stress. In contrast to WRN, BLM helicase defective in Bloom''s syndrome failed to rescue rad50 MMS sensitivity, but partially restored IR resistance, suggesting a delineation of function by the human RecQ helicases. [Copyright &y& Elsevier]

Abstract: The importance of helicases in nucleic acid metabolism and human disease has raised the bar for understanding how these unique enzymes function to perform their biological roles at the molecular level. Here we will describe experimental procedures and strategies to investigate the functions of helicases. These functional assays have been used to study DNA helicases important for the maintenance of genomic stability and genetically linked to age-related diseases and cancer. We will focus on the description of fluorometric helicase assays, protein displacement assays, and methods to characterize helicase activity on alternate DNA structures (triplex and quadruplex) used by our laboratory. The procedures to study these helicase functions are described in step-by-step detail to enable researchers interested in nucleic acid metabolism and related fields to apply these techniques to their own research questions. [Copyright &y& Elsevier]