Evidence for a Functional Monomeric Form of the Bacteriophage T4 Dda Helicase

RecQ family helicases play a key role in chromosome maintenance. Despite extensive biochemical, biophysical, and structural studies, the mechanism by which helicase unwinds double-stranded DNA remains to be elucidated. Using a wide array of biochemical and biophysical approaches, we have previously shown that the Escherichia coli RecQ helicase functions as a monomer. In this study, we have further characterized the kinetic mechanism of the RecQ-catalyzed unwinding of duplex DNA using the fluorometric stopped-flow method based on fluorescence resonance energy transfer. Our results show that RecQ helicase binds preferentially to 3-flanking duplex DNA. Under the pre-steady-state conditions, the burst amplitude reveals a 1:1 ratio between RecQ and DNA substrate, suggesting that an active monomeric form of RecQ helicase is involved in the catalysis. Under the single-turnover conditions, the RecQ-catalyzed unwinding is independent of the 3-tail length, indicating that functional interactions between RecQ molecules are not implicated in the DNA unwinding. It was further determined that RecQ unwinds DNA rapidly with a step size of 4 bp and a rate of ϳ21 steps/s. These kinetic results not only further support our previous conclusion that E. coli RecQ functions as a monomer but also suggest that some of the Superfamily 2 helicases may function through an "inchworm" mechanism.Helicases are molecular motor proteins that use the energy of nucleotide triphosphate hydrolysis to translocate along and separate the complementary strands of a nucleic acid duplex. These enzymes play essential roles in most aspects of the DNA metabolic pathway, such as replication, repair, recombination, and transcription (1-5). A large number of helicases have been identified; however, the mechanisms by which helicases unwind double-stranded DNA (dsDNA) 3 remain obscure.One of the major concerns in studying the unwinding mechanism of a DNA helicase is its oligomeric state. This is because an oligomeric structure could provide multiple potential binding sites for the DNA substrate and nucleotide cofactors, which are absolutely required for the helicase to translocate along the DNA track during the processive DNA unwinding. Indeed, the biochemical and structural studies have shown that some helicases assemble into stable cooperative hexameric rings (3). These helicases include the Escherichia coli DnaB (6, 7) and Rho (8, 9), bacteriophage T4 gp41 (10, 11), and T7 gp4 (12)(13)(14). DNA passes through such a central channel and is unwound with a high processivity. For the non-ring helicases, the enzymes could be assembled into oligomers, and the DNA binding sites may be located on the separate subunits. On the basis of quantitative analyses of DNA binding properties, a "rolling model" was proposed to explain factorial DNA unwinding (15). This model suggests that each monomer of the Rep dimer binds alternatively to ssDNA and dsDNA. This process is regulated by repeated binding and hydrolysis of ATP and release of ADP. In this way, Rep dimer rolls along ...

mentioning

confidence: 52%

Escherichia coli RecQ Is a Rapid, Efficient, and Monomeric Helicase

Zhang

Dou

Xie

et al. 2006

“…Oligonucleotides and Antibodies-All oligonucleotides were purchased from Integrated DNA Technologies and purified (103). The sequences used were: Cy3-G4DNA (5Ј-T 15 GAG GGT GGG TAG GGT GGG TAA-Cy3-3Ј), a well characterized G4DNA-forming sequence based on a region of the c-MYC promoter (104); Cy3-T 20 (5Ј-T 20 -Cy3-3Ј); Cy3-Scr (5Ј-TGT TGT TGT TGT TGT TGT TGT TGA GTG TAG TTG AAG-Cy3-3Ј), a scrambled version of the G4DNA sequence; Cy3-PS-Scr (5Ј-T*G*T TGT TGT TGT TGT TGT TGT TGA GTG TAG TTG A*A*G -Cy3-3Ј), where the asterisk indicates a phosphorothioate linkage; 3Ј-Bio-G4DNA (5Ј-T 15 GAG GGT GGG TAG GGT GGG TAA-BioTEG-3Ј); 3Ј-Bio-ssDNA (5Ј-T 15 -BioTEG-3Ј); Cy3-Cy5-G4DNA (5Ј-Cy5-T 15 GAG GGT GGG TAG GGT GGG TAA-Cy3-3Ј); and Cy3-Cy5-Scr (5Ј-Cy5-TGT TGT TGT TGT TGT TGT TGT TGA GTG TAG TTG AAG-Cy3-3Ј).…”

Section: Methodsmentioning

confidence: 99%

Evidence That G-quadruplex DNA Accumulates in the Cytoplasm and Participates in Stress Granule Assembly in Response to Oxidative Stress

Byrd¹,

Zybailov

Maddukuri³

et al. 2016

Self Cite

Cells engage numerous signaling pathways in response to oxidative stress that together repair macromolecular damage or direct the cell toward apoptosis. As a result of DNA damage, mitochondrial DNA or nuclear DNA has been shown to enter the cytoplasm where it binds to "DNA sensors," which in turn initiate signaling cascades. Here we report data that support a novel signaling pathway in response to oxidative stress mediated by specific guanine-rich sequences that can fold into G-quadruplex DNA (G4DNA). In response to oxidative stress, we demonstrate that sequences capable of forming G4DNA appear at increasing levels in the cytoplasm and participate in assembly of stress granules. Identified proteins that bind to endogenous G4DNA in the cytoplasm are known to modulate mRNA translation and participate in stress granule formation. Consistent with these findings, stress granule formation is known to regulate mRNA translation during oxidative stress. We propose a signaling pathway whereby cells can rapidly respond to DNA damage caused by oxidative stress. Guanine-rich sequences that are excised from damaged genomic DNA are proposed to enter the cytoplasm where they can regulate translation through stress granule formation. This newly proposed role for G4DNA provides an additional molecular explanation for why such sequences are prevalent in the human genome.

“…The simplest explanation is that gp32-dda interactions induce an active form of the helicase. dda alone does not form stable quaternary structures in solution (48), and there is strong evidence that monomeric dda can unwind model DNA substrates (49). One possibility is that an oligomeric form of dda is required for strand displacement synthesis reactions and that gp32 binding triggers this oligomerization.…”

Section: Relationship Between Equilibrium and Kinetic Properties Of Gmentioning

confidence: 99%

Dual Functions of Single-stranded DNA-binding Protein in Helicase Loading at the Bacteriophage T4 DNA Replication Fork

Wang

Villemain

et al. 2004

Semi-conservative DNA synthesis reactions catalyzed by the bacteriophage T4 DNA polymerase holoenzyme are initiated by a strand displacement mechanism requiring gp32, the T4 single-stranded DNA (ssDNA)-binding protein, to sequester the displaced strand. After initiation, DNA helicase acquisition by the nascent replication fork leads to a dramatic increase in the rate and processivity of leading strand DNA synthesis. In vitro studies have established that either of two T4-encoded DNA helicases, gp41 or dda, is capable of stimulating strand displacement synthesis. The acquisition of either helicase by the nascent replication fork is modulated by other protein components of the fork including gp32 and, in the case of the gp41 helicase, its mediator/ loading protein gp59. Here, we examine the relationships between gp32 and the gp41/gp59 and dda helicase systems, respectively, during T4 replication using altered forms of gp32 defective in either protein-protein or protein-ssDNA interactions. We show that optimal stimulation of DNA synthesis by gp41/gp59 helicase requires gp32-gp59 interactions and is strongly dependent on the stability of ssDNA binding by gp32. Fluorescence assays demonstrate that gp59 binds stoichiometrically to forked DNA molecules; however, gp59-forked DNA complexes are destabilized via protein-protein interactions with the C-terminal "A-domain" fragment of gp32. These and previously published results suggest a model in which a mobile gp59-gp32 cluster bound to lagging strand ssDNA is the target for gp41 helicase assembly. In contrast, stimulation of DNA synthesis by dda helicase requires direct gp32-dda protein-protein interactions and is relatively unaffected by mutations in gp32 that destabilize its ssDNA binding activity. The latter data support a model in which protein-protein interactions with gp32 maintain dda in a proper active state for translocation at the replication fork. The relationship between dda and gp32 proteins in T4 replication appears similar to the relationship observed between the UL9 helicase and ICP8 ssDNA-binding protein in herpesvirus replication.DNA helicase acquisition by a nascent DNA replication fork is essential for reconstituting rapid, processive DNA synthesis. This principle is illustrated dramatically by the bacteriophage T4 DNA replication system (1-2). T4 encodes two DNA helicases known to affect the movement and properties of DNA replication forks; the gene 41 protein (gp41) and the dda protein, respectively. gp41 is the essential replicative helicase of the T4 phage. This hexameric enzyme translocates processively in a 5Ј 3 3Ј direction on the displaced lagging strand of the dsDNA 1 template, thus enhancing the rate and processivity of leading strand DNA synthesis catalyzed by the T4 DNA polymerase holoenzyme (gp43, gp44/62, and gp45 proteins) (1-3). In addition, gp41 is an obligatory component of the T4 primosome (helicase/primase complex) and is, thus, essential for lagging strand DNA synthesis (4 -5). A second T4-encoded DNA helicase, dda protein, stimula...