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Home >> Molecular Biology Dictionary > DNA Helicases
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DNA helicases
In all cellular organisms from bacteria to h information is locked within a double helix for antiparallel deoxyribonucleic acid (DNA) strands. Although double-stranded DNA (dsDNA) is the form most suitable for secure information storage, hydrogen bonds formed between complementary bases (Watson-Crick base paring) impair readout of this information by the cellular machinery, which frequently requires a single-stranded D intermediate as a template.
The unwinding of dsDNA into ssDNA, a function critical for virtually very aspect of cellular DNA metabolism from DNA replication to homologous DNA recombination, is provided by a ubiquitous class of enzymes called DNA helicases. First identified in the 1970, DNA helicases are motor proteins that convert chemical energy into mechanical work. Chemical energy is derived from the hydrolysis of adenosine triphosphate (ATP) or other nucleoside triphosphates, and is coupled with mechanical work during at least two important steps within the helicase reaction cycle
(Fig. 1): (1) the unidirectional translocations along the substrate molecule and (2) the melting of the DNA duplex, which together result in the formation the ssDNA inter mediates essential for vital cellular processes.
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Schematic Representation of the Helicase Reaction
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Classifications
Helicases are divided into five main superfamilies based on the presence and composition of conserved amino acid motifs (often referred to as the helicase signature motifs). (It is important to note, however, that only a small fraction of these putative helicases have been studied biochemically and, of those proteins, not all have been shown to possess nucleic acid strand separation activity.)
Biochemical and structural data have suggested that helicases function as monomers, dimers, and multimers (predominantly hexamers) and that they can also be classified based on a substrate requirement for dsDNA, dsRNA, or DNA-RNA hybrids.
To unwind dsDNA efficiently, many DNA helicases need to initiate from an ssDNA region adjacent to the duplex part of the substrate molecule. Based on the requirement for an ssDNA overhang of a certain polarity, helicases are divided into two functional groups: those that utilize a 3'-terminated ssDNA are designated as 3' 5' helicases, whereas enzymes that require a 5' overhang are designated as 5' 3' helicases.
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Directional translocation
It is now generally believed that the observed polarity requirement of helicases is a consequence of a directional bias in translocation on ssDNA.
For example, the enzyme depicted in Fig. 1 is a 3' 5' helicase. Upon binding to the ssDNA, it starts moving toward the 5' end of the loading strand, which brings the enzyme to the ssDNA-dsDNA junction and subsequently through the duplex portion of the substrate It was shown recently that the rate of movement of the replication machinery at the fork is coordinated by an interaction between Dab and DNA polymerase (enzyme that synthesizes a daughter strand of DNA residues) that is educated by the subunit of the DNA polymerase.
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Then sub it bridges the polymerase dimer and the hexameric helicase, ducting a conformational change in DnaB that enhances its translocation rate by almost 3D-fold to 1000 base pairs per second. In the absence of, the replication machinery is uncoupled, and the polymerase simply follows DnaB as it unwinds DN at approximately 35 bp / s.
Single-molecule translocation visualization
Until recently, all biochemical data on helicases were derived from conventional bulk-phase techniques, which observe the population-averaged properties of large molecular ensembles. In 2001 two new approaches to visualize translocation by a single molecule of a helicase were reported. These new techniques successfully visualized translocation of a single molecule of RecBCD, a multifunctional heterotrimeric enzyme employed by E. coli to initiate homologous recombination at dsDNA breaks.
RecBCD is an exceptionally fast helicase that is furnished with all of the processivity and accessory factors it requires. The enzyme has a high affinity for blunt or nearly blunt dsDNA ends, and it can unwind, on average, 30,000 bp of dsDNA per binding event at a rate of 1000 bp / s, while simultaneously degrading the ssDNA products of its helicase activity.
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Optical trap visualization
In one approach, a device called an optical trap was used to manipulate individual, fluorescently labeled DNA molecules and to visualize their unwinding and degradation by the RecBCD enzyme (Fig. 2a). A dsDNA molecule, biotinylated at one end, was attached to streptavidin-coated polystyrene beads. The RecBCD enzyme was then prebound to the free DNA end in the absence of A TP. The bead was caught and held by lasers (the optical trap); buffer flowing through the optical cell caused the DNA to stretch out behind the trapped bead. The dsDNA was visualized by staining with a fluorescent intercalating dye (YOYO-1) and .appeared as a bright 15micrometer rod. Upon addition of ATP, the RecBCD enzyme mediated the unwinding of dsDNA, which was observed as a progressive shortening of the fluorescently labeled DNA molecule (Fig. 2b).
Tethered particle motion visualization
An alternative single-molecule approach used light microscopy to follow translocation of a biotin-tagged RecBCD enzyme bound to a streptavidin-coated polystyrene bead. In the tethered particle motion experiment (Fig. 3), dsDNA molecules, modified with digoxigenin at one end, were attached to a glass surface coated with antidigoxigenin antibodies. Bead-labeled RecBCD molecules were bound to the free (unmodified) dsDNA ends. Because the DNA acts as a flexible tether, RecBCD translocation was observed as a decrease in the Brownian motion (the irregular motion of small particles caused by the random bombardment by molecules in the surrounding medium) of the bead as it was pulled toward the glass surface.
Optical Trapping for studing RecBCD helicase nuclease |
Upon addtions of ATP and Unwinding continues until the helicase |
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Frames from a movie of DNA |
Optical Trap Apparatus |
Two Single Molecular Experiments |
Combined observations
The two single-molecule experiments are different yet complementary: the tethered particle motion experiment directly measures translocation, whereas the optical trap method (and conventioa1 bulk a says) measures dsDNA unwinding. Therefore, together, the studies provide additional powerful evidence for the coupling of DNA strand separation with movement of the helicase protein on its substrate lattice. Both single-molecule visualizations methods show that RecBCD translocates unidirectionally. and processively on dsDNA, with each molecule moving at a constant rate (within the limit of experimental detection). Although the average translocation rate is similar to that derived from bulk measurements, considerable variation is observed in the translocation rate of individual RecBCD enzymes This surprising observation is an example of the kind of formation that is accessible only by single-molecule studies.
Light Microscopy
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