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Current Opinion in Structural Biology Jun 2010Helicases of the superfamily (SF) 1 and 2 are involved in virtually all aspects of RNA and DNA metabolism. SF1 and SF2 helicases share a catalytic core with high... (Review)
Review
Helicases of the superfamily (SF) 1 and 2 are involved in virtually all aspects of RNA and DNA metabolism. SF1 and SF2 helicases share a catalytic core with high structural similarity, but different enzymes even within each SF perform a wide spectrum of distinct functions on diverse substrates. To rationalize similarities and differences between these helicases, we outline a classification based on protein families that are characterized by typical sequence, structural, and mechanistic features. This classification complements and extends existing SF1 and SF2 helicase categorizations and highlights major structural and functional themes for these proteins. We discuss recent data in the context of this unifying view of SF1 and SF2 helicases.
Topics: Amino Acid Sequence; Animals; DNA Helicases; Humans; Molecular Sequence Data; Protein Structure, Tertiary; RNA Helicases
PubMed: 20456941
DOI: 10.1016/j.sbi.2010.03.011 -
Nature Communications Oct 2022Polymerase Chain Reaction (PCR) is an essential method in molecular diagnostics and life sciences. PCR requires thermal cycling for heating the DNA for strand separation...
Polymerase Chain Reaction (PCR) is an essential method in molecular diagnostics and life sciences. PCR requires thermal cycling for heating the DNA for strand separation and cooling it for replication. The process uses a specialized hardware and exposes biomolecules to temperatures above 95 °C. Here, we engineer a PcrA M6 helicase with enhanced speed and processivity to replace the heating step by enzymatic DNA unwinding while retaining desired PCR characteristics. We name this isothermal amplification method SHARP (SSB-Helicase Assisted Rapid PCR) because it uses the engineered helicase and single-stranded DNA binding protein (SSB) in addition to standard PCR reagents. SHARP can generate amplicons with lengths of up to 6000 base pairs. SHARP can produce functional DNA, a plasmid that imparts cells with antibiotic resistance, and can amplify specific fragments from genomic DNA of human cells. We further use SHARP to assess the outcome of CRISPR-Cas9 editing at endogenous genomic sites.
Topics: Humans; DNA Helicases; Nucleic Acid Amplification Techniques; DNA-Binding Proteins; DNA; Polymerase Chain Reaction
PubMed: 36274095
DOI: 10.1038/s41467-022-34076-0 -
Molecular Cell Jan 2023Endogenous and exogenous agents generate DNA-protein crosslinks (DPCs), whose replication-dependent degradation by the SPRTN protease suppresses aging and liver cancer....
Endogenous and exogenous agents generate DNA-protein crosslinks (DPCs), whose replication-dependent degradation by the SPRTN protease suppresses aging and liver cancer. SPRTN is activated after the replicative CMG helicase bypasses a DPC and polymerase extends the nascent strand to the adduct. Here, we identify a role for the 5'-to-3' helicase FANCJ in DPC repair. In addition to supporting CMG bypass, FANCJ is essential for SPRTN activation. FANCJ binds ssDNA downstream of the DPC and uses its ATPase activity to unfold the protein adduct, which exposes the underlying DNA and enables cleavage of the adduct. FANCJ-dependent DPC unfolding is also essential for translesion DNA synthesis past DPCs that cannot be degraded. In summary, our results show that helicase-mediated protein unfolding enables multiple events in DPC repair.
Topics: DNA; DNA Damage; DNA Helicases; DNA Repair; DNA Replication; DNA-Binding Proteins; Protein Unfolding
PubMed: 36608669
DOI: 10.1016/j.molcel.2022.12.005 -
Methods (San Diego, Calif.) Oct 2016In this special Methods collection on DNA helicases, I have solicited articles from leading experts in the field with a priority to gather a defined series of papers on...
In this special Methods collection on DNA helicases, I have solicited articles from leading experts in the field with a priority to gather a defined series of papers on highly relevant topics that encompass biological, biochemical, and biophysical aspects of helicase function. The experimental approaches described provide an opportunity for both new and more experienced scientists to use the information for the design of their own investigations. The reader will find detailed methods for single-molecule studies, novel biochemical experiments, genetic analyses, and cell biological assays in a variety of systems with an emphasis placed on state-of-the-art techniques to measure helicase function. Contributing authors were strongly encouraged to provide a carefully constructed description of the methods employed so that others might use this information in a manner that will be useful for their own particular application and helicase of interest. This special issue of Methods dedicated to DNA helicases offers readers a treasure chest of unique experimental approaches and protocols focused on rapidly developing techniques that are useful for studying both in vivo and in vitro aspects of helicase function.
Topics: DNA; DNA Helicases
PubMed: 27565743
DOI: 10.1016/j.ymeth.2016.08.009 -
Biochemical Society Transactions Oct 2017Pif1 family helicases have multiple roles in the maintenance of nuclear and mitochondrial DNA in eukaryotes. Pif1 is involved in replication through barriers to... (Review)
Review
Pif1 family helicases have multiple roles in the maintenance of nuclear and mitochondrial DNA in eukaryotes. Pif1 is involved in replication through barriers to replication, such as G-quadruplexes and protein blocks, and reduces genetic instability at these sites. Another Pif1 family helicase in , Rrm3, assists in fork progression through replication fork barriers at the rDNA locus and tRNA genes. ScPif1 ( Pif1) also negatively regulates telomerase, facilitates Okazaki fragment processing, and acts with polymerase δ in break-induced repair. Recent crystal structures of bacterial Pif1 helicases and the helicase domain of human PIF1 combined with several biochemical and biological studies on the activities of Pif1 helicases have increased our understanding of the function of these proteins. This review article focuses on these structures and the mechanism(s) proposed for Pif1's various activities on DNA.
Topics: Bacteria; Crystallography, X-Ray; DNA; DNA Helicases; DNA Replication; G-Quadruplexes; Humans; Models, Molecular; Protein Structure, Quaternary; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 28900015
DOI: 10.1042/BST20170096 -
Viruses Aug 2021Bacteriophages have long been model systems to study the molecular mechanisms of DNA replication. During DNA replication, a DNA helicase and a DNA polymerase... (Review)
Review
Bacteriophages have long been model systems to study the molecular mechanisms of DNA replication. During DNA replication, a DNA helicase and a DNA polymerase cooperatively unwind the parental DNA. By surveying recent data from three bacteriophage replication systems, we summarized the mechanistic basis of DNA replication by helicases and polymerases. Kinetic data have suggested that a polymerase or a helicase alone is a passive motor that is sensitive to the base-pairing energy of the DNA. When coupled together, the helicase-polymerase complex is able to unwind DNA actively. In bacteriophage T7, helicase and polymerase reside right at the replication fork where the parental DNA is separated into two daughter strands. The two motors pull the two daughter strands to opposite directions, while the polymerase provides a separation pin to split the fork. Although independently evolved and containing different replisome components, bacteriophage T4 replisome shares mechanistic features of Hel-Pol coupling that are similar to T7. Interestingly, in bacteriophages with a limited size of genome like Φ29, DNA polymerase itself can form a tunnel-like structure, which encircles the DNA template strand and facilitates strand displacement synthesis in the absence of a helicase. Studies on bacteriophage replication provide implications for the more complicated replication systems in bacteria, archaeal, and eukaryotic systems, as well as the RNA genome replication in RNA viruses.
Topics: Bacteriophage T4; Bacteriophage T7; DNA Helicases; DNA Replication; DNA, Viral; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Kinetics; Models, Molecular; Viral Proteins; Virus Replication
PubMed: 34578319
DOI: 10.3390/v13091739 -
Methods (San Diego, Calif.) Oct 2016The growing number of DNA helicases implicated in hereditary disorders and cancer indicates that this particular class of enzymes plays key roles in genomic stability... (Review)
Review
The growing number of DNA helicases implicated in hereditary disorders and cancer indicates that this particular class of enzymes plays key roles in genomic stability and cellular homeostasis. Indeed, a large body of work has provided molecular and cellular evidence that helicases act upon a variety of nucleic acid substrates and interact with numerous proteins to enact their functions in replication, DNA repair, recombination, and transcription. Understanding how helicases operate in unique and overlapping pathways is a great challenge to researchers. In this review, we describe a series of experimental approaches and methodologies to identify and characterize DNA helicase inhibitors which collectively provide an alternative and useful strategy to explore their biological significance in cell-based systems. These procedures were used in the discovery of biologically active compounds that inhibited the DNA unwinding function catalyzed by the human WRN helicase-nuclease defective in the premature aging disorder Werner syndrome. We describe in vitro and in vivo experimental approaches to characterize helicase inhibitors with WRN as the model, anticipating that these approaches may be extrapolated to other DNA helicases, particularly those implicated in DNA repair and/or the replication stress response.
Topics: Biological Assay; DNA Helicases; DNA Repair; DNA Replication; Enzyme Inhibitors; Humans; Substrate Specificity
PubMed: 27064001
DOI: 10.1016/j.ymeth.2016.04.007 -
Nucleic Acids Research May 2012Conserved Iron-Sulfur (Fe-S) clusters are found in a growing family of metalloproteins that are implicated in prokaryotic and eukaryotic DNA replication and repair.... (Review)
Review
Conserved Iron-Sulfur (Fe-S) clusters are found in a growing family of metalloproteins that are implicated in prokaryotic and eukaryotic DNA replication and repair. Among these are DNA helicase and helicase-nuclease enzymes that preserve chromosomal stability and are genetically linked to diseases characterized by DNA repair defects and/or a poor response to replication stress. Insight to the structural and functional importance of the conserved Fe-S domain in DNA helicases has been gleaned from structural studies of the purified proteins and characterization of Fe-S cluster site-directed mutants. In this review, we will provide a current perspective of what is known about the Fe-S cluster helicases, with an emphasis on how the conserved redox active domain may facilitate mechanistic aspects of helicase function. We will discuss testable models for how the conserved Fe-S cluster might operate in helicase and helicase-nuclease enzymes to conduct their specialized functions that help to preserve the integrity of the genome.
Topics: Amino Acid Sequence; DNA; DNA Glycosylases; DNA Helicases; DNA Primase; Deoxyribonucleases; Iron-Sulfur Proteins; Molecular Sequence Data; Protein Structure, Tertiary
PubMed: 22287629
DOI: 10.1093/nar/gks039 -
Current Genetics Aug 2017Approximately, 1% of the genes in eukaryotic genomes encode for helicases, which make the number of helicases expressed in the cell considerably high. Helicases are... (Review)
Review
Approximately, 1% of the genes in eukaryotic genomes encode for helicases, which make the number of helicases expressed in the cell considerably high. Helicases are motor proteins that participate in many central aspects of the nuclear and mitochondrial genomes, and based on their helicase motif conservation, they are divided into different helicase families. The Pif1 family of helicases is an evolutionarily conserved helicase family that is associated with familial breast cancer in humans. The Schizosaccharomyces pombe Pfh1 helicase belongs to the Pif1 helicase family and is a multi-tasking helicase that is important for replication fork progression through natural fork barriers, for G-quadruplex unwinding, and for Okazaki fragment maturation, and these activities are potentially shared by the human Pif1 helicase. This review discusses the known functions of the Pfh1 helicase, the study of which has led to a better understanding of nucleic acid metabolism in eukaryotes.
Topics: Breast Neoplasms; DNA Helicases; DNA Replication; Eukaryotic Cells; Female; G-Quadruplexes; Humans; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 28054200
DOI: 10.1007/s00294-016-0675-2 -
Methods (San Diego, Calif.) Aug 2022DDX43 (DEAD-box helicase 43), also known as HAGE (helicase antigen gene), is a member of the DEAD-box protein family. It contains a K homology (KH) domain in its N...
DDX43 (DEAD-box helicase 43), also known as HAGE (helicase antigen gene), is a member of the DEAD-box protein family. It contains a K homology (KH) domain in its N terminus, a helicase core domain in its C terminus, and a flexible linker domain in between. DDX43 expression is low or undetectable in normal tissue, but is overexpressed in many tumors; therefore, it is considered a potential target molecule for cancer therapy. We, along with other groups, have shown that DDX43 is an ATP-dependent RNA and DNA helicase, and the KH domain is required for its ATPase and unwinding activity. Electrophoretic mobility shift assay (EMSA), SELEX (systematic evolution of ligands by exponential enrichment), chromatin immunoprecipitation (ChIP)-seq, crosslinking immunoprecipitation (CLIP)-seq, and nuclear magnetic resonance (NMR) showed that the KH domain prefers to bind pyrimidine-rich ssDNA and ssRNA, such as TTGT in the promoter regions of genes. Moreover, the KH domain facilitates the substrate specificity and processivity of the DDX43 helicase. No animal model has been generated for DDX43; cellular studies have revealed that DDX43 has roles in piRNA amplification, tumorigenesis, RAS signaling, and innate immunity. Structural and functional studies of DDX43 will not only advance our understanding of DEAD-box helicases and KH domains, but also shed light on the application of DDX43 as therapeutics, where its key binding sites can be targeted by small molecules and natural products as an alternative approach in treating DDX43 overexpressed cancers.
Topics: Binding Sites; DEAD-box RNA Helicases; DNA Helicases; RNA; Substrate Specificity
PubMed: 35257897
DOI: 10.1016/j.ymeth.2022.03.002