DNA Helicase

Type I DNA restriction enzymes are large, molecular machines possessing DNA methyltransferase, ATPase, DNA translocase and endonuclease activities. The ATPase, DNA translocase and endonuclease activities are speci®ed by the restriction (R) subunit of the enzyme. We demonstrate that the R subunit of the EcoKI type I restriction enzyme comprises several different functional domains. An N-terminal domain contains an amino acid motif identical with that forming the catalytic site in simple restriction endonucleases, and changes within this motif lead to a loss of nuclease activity and abolish the restriction reaction. The central part of the R subunit contains amino acid sequences characteristic of DNA helicases. We demonstrate, using limited proteolysis of this subunit, that the helicase motifs are contained in two domains. Secondary structure prediction of these domains suggests a structure that is the same as the catalytic domains of DNA helicases of known structure. The C-terminal region of the R subunit can be removed by elastase treatment leaving a large fragment, stable in the presence of ATP, which can no longer bind to the other subunits of EcoKI suggesting that this domain is required for protein assembly. Considering these results and previous models of the methyltransferase part of these enzymes, a structural and operational model of a type I DNA restriction enzyme is presented.

DNA polymerase theta (Polq) has been identified as a crucial alternative non-homologous end-joining fac- tor in mammalian cells. Polq is upregulated in a range of cancer cell types defective in homologous recom- bination, and knockdown has been shown to inhibit cell survival in a subset of these, making it an attrac- tive target for cancer treatment. We present crystal structures of the helicase domain of human Polq in the presence and absence of bound nucleotides, and a characterization of its DNA-binding and DNA- stimulated ATPase activities. Comparisons with related helicases from the Hel308 family identify several unique features. Polq exists as a tetramer both in the crystals and in solution. We propose a model for DNA binding to the Polq helicase domain in the context of the Polq tetramer, which suggests a role for the helicase domain in strand annealing of DNA templates for subsequent processing by the polymerase domain.

The packaging of the eukaryotic genome in chromatin presents barriers that restrict the access of enzymes that process DNA 1,2 . To overcome these barriers, cells possess a number of multi-protein, ATP-dependent chromatin remodelling complexes, each containing an ATPase subunit from the SNF2/SWI2 superfamily 3,4 . Chromatin remodelling complexes function by increasing nucleosome mobility and are clearly implicated in transcription 5±7 . Here we have analysed SNF2/SWI2-and ISWI-related proteins to identify remodelling complexes that potentially assist other DNA transactions. We puri®ed a complex from Saccharomyces cerevisiae that contains the Ino80 ATPase 8 . The INO80 complex contains about 12 polypeptides including two proteins related to the bacterial RuvB DNA helicase 9±11 , which catalyses branch migration of Holliday junctions. The puri®ed complex remodels chromatin, facilitates transcription in vitro and displays 39 to 59 DNA helicase activity. Mutants of ino80 show hypersensitivity to agents that cause DNA damage, in addition to defects in transcription 8 . These results indicate that chromatin remodelling driven by the Ino80 ATPase may be connected to transcription as well as DNA damage repair.

Helicases, enzymes that unwind DNA or RNA structure, are present in the cell nucleus and in the mitochondrion. Although the majority of the helicases unwind DNA or RNA duplexes, some of these proteins are known to resolve unusual structures such as Gquadruplexes (G4) in vitro. G4 may form stable barrier to the progression of molecular motors tracking on DNA. Monitoring G4 unwinding by these enzymes may reveal the mechanisms of the enzymes and provides information about the stability of these structures. In the experiments presented herein, we developed a reliable, inexpensive and rapid fluorescencebased technique to monitor the activity of G4 helicases in real time in a 96-well plate format. This system was used to screen a series of G4 structures and G4 binders for their effect on the Pif1 enzyme, a 5 to 3 DNA helicase. This simple assay should be adaptable to analysis of other helicases and G4 structures.

DNA helicases are ubiquitous molecular motor proteins which harness the chemical free energy of ATP hydrolysis to catalyze the unwinding of energetically stable duplex DNA, and thus play important roles in nearly all aspects of nucleic acid metabolism, including replication, repair, recombination, and transcription. They break the hydrogen bonds between the duplex helix and move unidirectionally along the bound strand. All helicases are also translocases and DNA-dependent ATPases. Most contain conserved helicase motifs that act as an engine to power DNA unwinding. All DNA helicases share some common properties, including nucleic acid binding, NTP binding and hydrolysis, and unwinding of duplex DNA in the 3¢ to 5¢ or 5¢ to 3¢ direction. The minichromosome maintenance (Mcm) protein complex (Mcm4/6/7) provides a DNA-unwinding function at the origin of replication in all eukaryotes and may act as a licensing factor for DNA replication. The RecQ family of helicases is highly conserved from bacteria to humans and is required for the maintenance of genome integrity. They have also been implicated in a variety of human genetic disorders. Since the discovery of the first DNA helicase in Escherichia coli in 1976, and the first eukaryotic one in the lily in 1978, a large number of these enzymes have been isolated from both prokaryotic and eukaryotic systems, and the number is still growing. In this review we cover the historical background of DNA helicases, helicase assays, biochemical properties, prokaryotic and eukaryotic DNA helicases including Mcm proteins and the RecQ family of helicases. The properties of most of the known DNA helicases from prokaryotic and eukaryotic systems, including viruses and bacteriophages, are summarized in tables.

Guanine-rich DNA strands can fold in vitro into non-canonical DNA structures called G-quadruplexes. These structures may be very stable under physiological conditions. Evidence suggests that G-quadruplex structures may act as 'knots' within ge-nomic DNA, and it has been hypothesized that proteins may have evolved to remove these structures. The first indication of how G-quadruplex structures could be unfolded enzymatically came in the late 1990s with reports that some well-known duplex DNA helicases resolved these structures in vitro. Since then, the number of studies reporting G-quadruplex DNA unfolding by helicase enzymes has rapidly increased. The present review aims to present a general overview of the helicase/G-quadruplex field.

RuvB Family of Enzymes belong to AAA+ Class Enzyme RuvB belongs to a category of proteins called the AAA+ class (ATPases associated with various cellular activities), which contains one or two copies of a well-conserved 230-to 250amino-acid cassette called the AAA module that encompasses the Walker A and B motifs. The activities associated with AAA modules are involved in diverse cellular functions. 17 Bacterial RuvB, yeast RuvBs (RuvB1 and RuvB2) and human RuvB1 and RuvB2 have been studied under different names. Alternate names

The HMI1 gene encodes a DNA helicase that localizes to the mitochondria and is required for maintenance of the mitochondrial DNA (mtDNA) genome of Saccharomyces cerevisiae. Identified based on its homology with E. coli uvrD, the HMI1 gene product, Hmi1p, has been presumed to be involved in the replication of the 80 kb linear S. cerevisiae mtDNA genome. Here we report the purification of Hmi1p to apparent homogeneity and provide a characterization of the helicase reaction and the ATPase reaction with regard to NTP preference, divalent cation preference and the stimulatory effects of different nucleic acids on Hmi1p-catalysed ATPase activity. Genetic complementation assays indicate that mitochondrial localization of Hmi1p is essential for its role in mtDNA metabolism. The helicase activity, however, is not essential. Point mutants that lack ATPase/helicase activity partially complement a strain lacking Hmi1p. We suggest several possible roles for Hmi1p in mtDNA metabolism.

DNA helicases have important roles in various DNA transactions, including DNA replication, recombination and repair, and thus their down regulation often results in genome instability. 2) Several human genetic disorders having accelerated tumorgenesis and aging are caused by mutations in genes encoding helicases, and the down regulation of helicase genes often causes cell death. 3) The Pif1 DNA helicase (Pif1p) was identified first in Saccharomyces cerevisiae 4) and was classified as a member of SFI helicase conserved from yeast to human. 5) The function of Pif1p is implicated in mtDNA repair, 6) rDNA replication 7-9) and in the regulation of telomere length. 10,11) Also Pif1p is thought to be a region-specific DNA helicase because it requires a specific DNA structure for the efficient DNA unwinding in vitro. 12) The yeast Pif1p was found to unwind DNA in a 5Ј-to-3Ј direction. 13) S. cerevisiae cells have two homologues of Pif1-like helicases, Pif1p itself and Rrm3p. The Rrm3p helicase is thought to solve the replication forks traversing protein-DNA complexes that occur in impediments in S phase progression. 9) The yeast cells lacking both Pif1 and Rrm3 helicase genes are viable although the mutant cells show elevated genome instability. 8) In contrast, Pfh1p, the Pif1-like helicase in Saccharomyces pombe, is essential for viability and it was speculated that Pfh1p plays important roles in the replication of chromosome DNA. 14,15) Thus, although Pif1-like helicases exist in many organisms, most of studies have been made with yeast species, and little is known for Pif1 helicase in mammalian cells. Recently, Zhang et al. reported that the human Pif1 in the nucleus participated in the maintenance of telomere length. 16) In this paper, we describe the human Pif1 gene, and characterize two isoforms of gene products for the first time which are generated by the alternative splicing, focusing on their differential subcellular localization. MATERIALS AND METHODS Cell Culture and Transfection HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM;

Eukaryotic replication initiation is highly regulated and dynamic. It begins with the origin recognition complex (ORC) binding DNA sites called origins of replication. ORC, together with Cdc6 and Cdt1, mediate pre-replicative complex (pre-RC) assembly by loading a double hexamer of Mcm2–7: the core of the replicative helicase. Here, we use single-mole- cule imaging to directly visualize Saccharomyces cerevisiae pre-RC assembly and replisome firing in real time. We show that ORC can locate and stably bind origins within large tracts of non-origin DNA and that Cdc6 drives ordered pre-RC assembly. We further show that the dynamics of the ORC-Cdc6 interaction dictate Mcm2–7 loading specificity and that Mcm2–7 double hexamers form preferentially at a native origin sequence. Finally, we demonstrate that single Mcm2–7 hexamers propagate bidirection- ally, monotonically, and processively as constituents of active replisomes.

Two new human DNA helicase genes, RecQ4 and RecQ5, that belong to the RecQ helicase family were cloned and characterized. The addition of these genes increases the total to five helicase genes in the human RecQ family, which includes helicases involved in Bloom and Werner syndromes, the genetic diseases manifesting the distinctive but overlapping clinical phenotypes of immunodeficiency, premature aging, and an enhanced risk of cancer. The RecQ4 helicase is as large as the Bloom (BLM) and Werner (WRN) helicases, and its gene expression profile is organ-specific, resembling that of BLM helicase. In contrast, the RecQ5 helicase has a low molecular weight, similar to the human progenitor RecQ1 helicase, and is expressed in all the organs examined. All five human helicase genes are expressed in cultured K562 leukemia and fibroblast cells. Synchronized K562 cell cultures showed that the genes RecQ4 and BLM, and RecQ1 and WRN, seem to be upregulated at the G1/S and G2/M phases, respectively, of the cell cycle. The biological significance of multiple species of human RecQ helicases, which are apparently nonessential for life but may be related to distinct diseases, is discussed in light of the fact that unicellular organisms, like Escherichia coli and yeast, contain only one species of helicase of this particular family.

MITF/TFE translocation renal cell carcinoma (TRCC) is a rare subtype of kidney cancer. Its incidence and the genome-wide characterization of its genetic origin have not been fully elucidated. We performed RNA and exome sequencing on an exploratory set of TRCC (n = 7), and validated our findings using The Cancer Genome Atlas (TCGA) clear-cell RCC (ccRCC) dataset (n = 460). Using the TCGA dataset, we identified seven TRCC (1.5%) cases and determined their genomic profile. We discovered three novel partners of MITF/TFE (LUC7L3, KHSRP, and KHDRBS2) that are involved in RNA splicing. TRCC displayed a unique gene expression signature as compared with other RCC types, and showed activation of MITF, the transforming growth factor β1 and the PI3K complex targets. Genes differentially spliced between TRCC and other RCC types were enriched for MITF and ID2 targets. Exome sequencing of TRCC revealed a distinct mutational spectrum as compared with ccRCC, with frequent mutations in chromatin-remo...

SUMMARYA plastid-like organelle, the apicoplast, is essential to the majority of medically and veterinary important apicomplexan protozoa including Toxoplasma gondii and Plasmodium. The apicoplast contains multiple copies of a 35 kb genome, the replication of which is dependent upon nuclear-encoded proteins that are imported into the organelle. In P. falciparum an unusual multi-functional gene, pfprex, was previously identified and inferred to encode a protein with DNA primase, DNA helicase and DNA polymerase activities. Herein, we report the presence of a prex orthologue in T. gondii. The protein is predicted to have a bi-partite apicoplast targeting sequence similar to that demonstrated on the PfPREX polypeptide, capable of delivering marker proteins to the apicoplast. Unlike the P. falciparum gene that is devoid of introns, the T. gondii prex gene carries 19 introns, which are spliced to produce a contiguous mRNA. Bacterial expression of the polymerase domain reveals the protein ...

Hepatitis C virus (HCV) nonstructural protein 3 (NS3) is a known RNA helicase, an enzyme that unwinds RNA x DNA and RNA x RNA duplexes. We have now deciphered the biochemical characteristics of the HCV NS3 DNA helicase activity. Recombinant NS3 was expressed in Escherichia coli, purified to near homogeneity, and tested for DNA helicase activity. The optimal conditions for DNA unwinding (for example, the preferred pH and magnesium ion concentration) were similar to those for RNA unwinding. The DNA helicase activity was very sensitive to potassium ion concentration, while DNA binding and DNA-stimulated ATPase activities were not. The direction of DNA unwinding was determined to be 3' to 5'. All four ribonucleoside triphosphates (ATP, GTP, CTP, UTP) and deoxynucleoside triphosphates (dATP, dGTP, dCTP, dTTP) could serve as energy sources, but GTP and dGTP were less efficient than the others. When nucleotide analog inhibitors were added to the DNA helicase reaction, the overall o...

Mini chromosome maintenance (MCM) proteins 2-7, a subgroup of the large AAA ATPase family are critically required for eukaryotic DNA replication. These proteins are most likely responsible for unwinding DNA at the replication forks. Besides this function, some MCMs are also involved in other chromosome transactions such as transcription, chromatin remodeling and genome stability. All the MCMs contain a conserved region of ~200 amino acids responsible for nucleotide binding. The importance of MCM proteins is evident by the fact that deregulation of the activity of MCM family of proteins appears to be directly linked to human carcinogenesis. This article will focus on members of this important family of proteins from the malaria parasite Plasmodium falciparum and their comparison with the human host.

Bloom syndrome (BS) is a rare autosomal recessive disorder characterized by growth deficiency, immunodeficiency, genomic instability, and the early development of cancers of many types. BLM, the protein encoded by BLM, the gene mutated in BS, is localized in nuclear foci and absent from BS cells. BLMencodes a DNA helicase, and proteins from three missense alleles lack displacement activity. BLM transfected into BS cells reduces the frequency of sister chromatid exchanges and restores BLM in the nucleus. Missense alleles fail to reduce the sister chromatid exchanges in transfected BS cells or restore the normal nuclear pattern. BLM complements a phenotype of aSaccharomyces cerevisiae sgs1 top3 strain, and the missense alleles do not. This work demonstrates the importance of the enzymatic activity of BLM for its function and nuclear localization pattern.

Helicases, enzymes that unwind DNA or RNA structure, are present in the cell nucleus and in the mitochondrion. Although the majority of the helicases unwind DNA or RNA duplexes, some of these proteins are known to resolve unusual structures such as G-quadruplexes (G4) in vitro. G4 may form stable barrier to the progression of molecular motors tracking on DNA. Monitoring G4 unwinding by these enzymes may reveal the mechanisms of the enzymes and provides information about the stability of these structures. In the experiments presented herein, we developed a reliable, inexpensive and rapid fluorescence-based technique to monitor the activity of G4 helicases in real time in a 96-well plate format. This system was used to screen a series of G4 structures and G4 binders for their effect on the Pif1 enzyme, a 5' to 3' DNA helicase. This simple assay should be adaptable to analysis of other helicases and G4 structures.