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20 August 2002 19:8 YB030940.tex McGraw Hill YB of Science & Technology Keystroked: 08/10/ Initial MS Page Sequence Stamp: 00334 Article Title: DNA helicases Article ID: YB 1st Classification Number: 370200 2nd Classification Number: Sequence Number:
Galley Proofs
DNA helicases 1
DNA helicases
In all cellular organisms from bacteria to humans, genetic information is locked within a double helix formed by the two antiparallel deoxyribonucleic acid (DNA) strands. Although double-stranded DNA (dsDNA) is the form most suitable for secure informa- tion storage, hydrogen bonds formed between com- plementary bases (Watson-Crick base pairing) impair readout of this information by the cellular machin- ery, which frequently requires a single-stranded DNA (ssDNA) intermediate as a template. The unwinding of dsDNA into ssDNA, a function critical for virtu- ally every aspect of cellular DNA metabolism from RNA synthesis to homologous DNA recombination, is provided by a ubiquitous class of enzymes called DNA helicases. First identified in the 1970s, DNA he- licases are motor proteins (often called DNA motors) 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 unidirec- tional translocations along the substrate molecule and (2) the melting of the DNA duplex, which together result in the formation of the ssDNA inter- Author: Is “which together result” okay? mediates essential for vital cellular processes. Classifications. Helicases are divided into five main superfamilies based on the presence and composi- tion of conserved amino acid (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
n(ATP)
n(ADP + Pi )
reannealingprevented reannealing
DNA-binding proteins
3´
3´ 5´
5´
helicase enzyme
Fig. 1. Schematic representation of the helicase reaction. The helicase enzyme translocates along the DNA molecule and separates the strands. Energy for this unfavorable reaction is provided by the hydrolysis of adenosine triphosphates (ATP) to adenosine diphosphates (ADP) and inorganic phosphate ions (Pi). In the presence of a single-stranded DNA binding protein, reannealing of the DNA duplex is prevented. The helicase depicted here displays a 3 ′^ → 5 ′^ polarity, tracking unidirectionally along the lower of the two DNA strands in the duplex (the loading strand).
Author: “loading strand” OK per text?
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2 DNA helicases
have suggested that helicases function as monomers, dimers, and multimers (predominantly hexamers) and that they can also be classified based on a sub- strate requirement for dsDNA, dsRNA, or DNA-RNA hybrids. To unwind dsDNA efficiently, many DNA helicases need to initiate from an ssDNA region ad- jacent to the duplex part of the substrate molecule. Based on the requirement for an ssDNA overhang of a certain polarity, helicases are further divided into two functional groups: those that utilize a 3′- terminated ssDNA are designated as 3′^ → 5 ′^ heli- cases, whereas enzymes that require a 5′^ overhang are designated as 5′^ → 3 ′^ helicases. Directional translocation. It is now generally belie- ved that the observed polarity requirement of heli- cases is a consequence of a directional bias in translo- cation 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 load- ing strand, which brings the enzyme to the ssDNA- dsDNA junction and subsequently through the du- plex portion of the substrate. Evidence for directional translocation on ssDNA was provided by two different approaches. The first examined the dependence of helicase ATPase activ- ity on the length of the ssDNA substrate; the second, based on the ability of many helicases to Author: Edit okay? create sufficient force during ssDNA translocation to disrupt the tight interaction between streptavidin and biotin ( K (^) d = 10 −^15 M), measured the ability of the helicase to increase the rate of streptavidin dis- sociation from DNA substrates biotinylated at either the 3′^ or 5′^ end. This second method was used suc- cessfully to determine the directionality of move- ment of several helicases on ssDNA. High-resolution structural data suggest that the helicase signature mo- tifs are not essential for the duplex DNA separation per se, but for the ATP-dependent unidirectional motion of the helicases on either single- or double- stranded DNA lattices. Consequently, it has been pro- posed that the helicase signature motifs define a mod- ular structure that functions as the DNA motor, while additional domains, which may vary from one pro- tein to another, might be responsible for the DNA unwinding. Accessory factors. Once dsDNA unwinding is achi- eved, spontaneous reannealing of the duplex may be avoided if the nascent ssDNA strands are trapped by single-stranded DNA binding proteins that hand off the intermediates to the next step in a reaction pathway (Fig. 1). Although ssDNA binding proteins have frequently been shown to stimulate helicase activity in vitro, helicase activity can also be stimu- lated by other accessory factors that increase the rate or processivity of unwinding. The primary replica- tive helicase of Escherchia coli , DnaB, is a good example of a helicase that acts poorly in isolation from the accessory factors with which the enzyme is intended to operate. As part of the replisome (the DNA synthesis machinery of the cell), the role of DnaB is to separate the DNA strands at the replication fork. However, it was shown recently that the rate of
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4 DNA helicases
Sample side (no ATP) Reaction side (+ATP)
(a)
ATP
ADP RecBCDunwinding
YOYO-
flow
RecBCD
YOYO-
flow
ATP
ADP
(1) (2) (3)
(1) (2) (3) (b) Fig. 2. Optical track visualization. ( a ) Optical trapping method for studying RecBCD helicase/nuclease at the single-molecule level. (1) A polystyrene bead is held in the optical trap with dsDNA (stained with the fluorescent dye YOYO-1) stretched out in the flow behind it. (2) Upon addition of ATP, the helicase begins to unwind and degrade the DNA. (3) Unwinding continues until the helicase reaches the bead or falls off of its DNA track. ( b ) Frames from a movie of DNA unwinding and degradation in the optical trap apparatus. The frames are equivalent to the representation in a****. ( The original movie of the helicase in action may be viewed in its entirety at http://microbiology.ucdavis.edu/sklab/kowalczykowskilab.htm )
Author: Okay to delete “RecBCD” from “complex” label in Fig. 2a?
tethered particle motion experiment directly mea- sures translocation, whereas the optical trap method (and conventional bulk assays) measures dsDNA un- winding. Therefore, together, the studies provide ad- ditional powerful evidence for the coupling of DNA strand separation with movement of the helicase pro- tein on its substrate lattice. Both single-molecule vi- sualization 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 information that is accessible only by single-molecule studies. Conclusion. In the last 10 years, considerable pro- gress has been made in the understanding of the molecular mechanisms of DNA helicases. Although many questions remain, perhaps the next challenge in this field is to understand how these DNA motors are incorporated into and used by large multipro- tein complexes, such as the replisome, to orchestrate complex DNA processing events.
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DNA helicases 5
extent of the bead Brownian motion
biotin-tagged RecBCD
streptavidin-coated bead
digoxigenin-tagged
dsDNA
anti-digoxigenin antibody
glass Fig. 3. Tethered particle motion experiment to study DNA translocation by single RecBCD helicase/nuclease molecules. A dsDNA molecule is attached to a glass surface, and RecBCD molecules are attached to polystyrene beads. As RecBCD tracks along the DNA molecule in an ATP-dependent manner, it gradually draws the bead closer to the glass surface. This translocation results in a decrease in the Brownian motion of the bead that can be measured by light microscopy. ( Adapted from http://www.bio.brandeis.edu/ ∼ gelles/movies.html )
For background information see ADENOSINE TRIPHOSPHATE (ATP); DEOXYRIBONUCLEIC ACID (DNA) ; ENZYME; MOLECULAR BIOLOGY; NUCLEOPRO- TEIN in the McGraw-Hill Encyclopedia of Science & Technology. Maria Spies; Mark S. Dillingham; Stephen C. Kowalczykowski Key Words: DNA Helicase, ATP hydrolysis, molecu- lar motors, DNA replication, RNA transcription, DNA recombination and repair Bibliography. B. Alberts et al., Molecular Biology of the Cell , 3d ed. (II, Chapter 6), Garland Publish- ing, New York, 1994; P. R. Bianco et al., Processive translocation and DNA unwinding by individual RecBCD enzyme molecules, Nature , 409(18):374– 378, 2001; K. M. Dohoney and J. Gelles, χ-Sequence recognition and DNA translocation by single RecBCD helicase/nuclease molecules, Nature , 409(18):370– 374, 2001; H. Lodish et al., Molecular Cell Biology , 4th ed. (Chap. 12), W H Freeman, New York, 2000; P. Soultanas and D. B. Wigley, Unwinding the “Gordian Knot” of helicase action, TIBS , 26(1):47– 54, 2001. Additional reading. D. A. Arnold and S. C. Kowalczykowski, RecBCD helicase/nuclease, in Encyclopedia of Life Sciences , Nature Publishing Group, London, 1999; C. Bustamante et al., The physics of molecular motors, Acc. Chem. Res ., 34:412–420, 2001; P. D. Morris et al., Biotin- streptavidin-labeled oligonucleotides as probes of he- licase mechanisms, METHODS , 23:149–159, 2001; P. H. von Hippel and E. Delagoutte, A general model for nucleic acid helicases and their “coupling” within macromolecular machines, Cell , 104(2):177–90, 2001; S. C. West, DNA helicases: New breeds of trans- locating motors and molecular pumps, Cell , 86:177– 180, 1996.
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