Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-30
2001-10-30
Wang, Andrew (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S007720, C435S007800, C435S091100, C435S183000, C536S023100
Reexamination Certificate
active
06309839
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of molecular biology. More specifically, the present invention relates to helicases and methods of screening for compounds that inhibit or stimulate helicase enzyme activity.
DESCRIPTION OF THE RELATED ART
DNA helicases are molecular motors that transduce the energy obtained from hydrolysis of nucleotide triphosphates (NTPs) to perform the mechanical work of unwinding double-stranded (ds) DNA (1,2,3,4,5). These enzymes are ubiquitous and necessary for most aspects of nucleic acid metabolism, including replication, repair, and recombination. Several disease states have recently been associated with defective helicases, such as Bloom's syndrome (6) and xerodoma pigmentosum (7). However, the biochemical mechanism(s) of helicases are largely unknown.
The
E. coli
Rep helicase has been closely studied and a mechanism has been proposed for this dimeric enzyme (2, 5). One subunit of Rep is proposed to bind ssDNA while the other binds and unwinds dsDNA in a cyclic process coordinated by binding and hydrolysis of ATP. The preferential affinity of one subunit of the dimer for dsDNA over ssDNA at a ds/ss DNA junction is proposed to drive translocation of the enzyme via a “rolling” or “subunit switching” mechanism. This mechanism predicts that little or no directional bias occurs during translocation on ssDNA substrates. In contrast, others have proposed that some helicases translocate unidirectionally on ssDNA, and that such activity is important to the overall mechanism of dsDNA unwinding (8). A model for translocation by the
E. coli
transcription termination protein Rho has been provided that includes a directionally biased random walk along RNA (9). Evidence has been provided suggesting that the bacteriophage helicases gp41and Dda translocate unidirectionally on ssDNA (10, 11).
gp41 subunits can oligomerize into a hexamer in the presence of ATPor GTP(12), and serves as the replicative helicase of bacteriophage T4. Investigators using electron microscopy have determined that some hexameric helicases bind to DNA by encircling it, such that the DNA passes through the central channel of the hexamer (reviewed in 3). Results from biochemical experiments (13) may be interpreted as being consistent with similar DNA binding by gp41. The present invention provides additional evidence based upon biochemical studies that suggests that gp41 encircles ssDNA.
One outstanding question regarding helicase function deals with the direction and mechanism of translocation on ssDNA. Studies of another molecular motor protein, the F
1
-ATPase, may provide clues towards answering this question. The hexameric form of helicases with DNA passing through the central channel is somewhat analogous to the F
1
-ATPase, which consists of a hexamer of &agr;3_&bgr; subunits encircling a &ggr; subunit (14). Rotation of the &ggr; subunit within the cylinder of the hexamer has been directly observed in the presence of ATP (15). Recently, the dTTPase activity of the hexameric gene 4 helicase from bacteriophage T7 was found to resemble the mechanism of the F
1
-ATPase, in which three of the six potential nucleotide binding sites on the hexamer are catalytic sites and three are noncatalytic sites (16). A rotational movement of the gene 4 hexamer around the ssDNA was proposed to result in unidirectional translocation and unwinding of duplex DNA.
Others have recently proposed an inch-worm mechanism involving unidirectional translocation on RNA or ssDNA based upon the crystal structure of the non-structural protein 3 (NS3) helicase from the hepatitis C virus (17). If helicases translocate unidirectionally on ssDNA, the enzyme may produce a force in the direction of translocation. The effect of force on biochemical kinetics has recently received much attention (18). Technical innovations with atomic force microscopy (AFM), optical tweezers, and other methods have allowed direct measurement of the forces involved in binding of a ligand to its receptor as well as the forces generated by a translocating enzyme (19). The invention described herein provides evidence for production of force by DNA helicases, which suggests that the helicase has a strong directional bias on ssDNA. When challenged with biotinylated-oligonucleotide substrates to which streptavidin has been bound, the gp41 and Dda helicases can rapidly displace the streptavidin, suggesting that these enzymes impart a strong unidirectional force on the streptavidin.
The prior art is deficient in methods of screening for compounds that inhibit or stimulate helicase enzyme activity that do not rely upon a double-stranded DNA template. The present invention fulfills this longstanding need and desire in the art.
SUMMARY OF THE INVENTION
The present invention describes a new assay for studying enzymatic activity of gp41, Dda, HCV NS3, SV40 T antigen, and any other helicase on single-stranded DNA using the rate of dissociation of streptavidin from various biotinylated oligonucleotides in the presence of a helicase. gp41, Dda HCV NS3, and SV40 T antigen were found to significantly enhance the dissociation rate of streptavidin from biotin-labeled oligonucleotides in an ATP dependent reaction, thereby indicating that these enzymes are capable of imparting a significant amount of force on a molecule blocking their path.
In one embodiment, the present invention provides for a method of screening for compounds that inhibit or stimulate helicase enzyme activity, comprising the steps of: (a) combining under appropriate conditions: (i) a helicase enzyme; and (ii) a biotinylated oligonucleotide bound to streptavidin (SA-B-oligo), thereby producing helicase-associated SA-B-oligo; (b) contacting a sample of the helicase-associated SA-B-oligo with a compound, thereby producing a compound-treated helicase-associated SA-B-oligo sample; and (c) measuring the amount of dissociation of the biotinylated oligonucleotide from streptavidin in the compound-treated helicase-associated SA-B-oligo sample and an untreated helicase-associated SA-B-oligo sample. Less dissociation of the biotinylated oligonucleotide from the streptavidin in the compound-treated helicase-associated SA-B-oligo sample than in the untreated helicase-associated SA-B-oligo sample indicates that the compound inhibits the helicase enzyme activity. Greater dissociation of the biotinylated oligonucleotide from the streptavidin in the compound-treated helicase-associated SA-B-oligo sample than in the untreated helicase-associated SA-B-oligo sample indicates that the compound stimulates the helicase enzyme activity.
In yet another embodiment of the present invention, there is provided a method of releasing a streptavidin-captured, biotinylated oligonucleotide, comprising the steps of: (a) contacting a streptavidin-captured biotinylated oligonucleotide with a helicase; and (b) collecting the biotinylated oligonucleotide released from the streptavidin.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.
REFERENCES:
Ahnert et al. J. Biol. Chem. vol. 272, No. 51, pp. 32,267-32,273, 1997.*
Young et al. J. Mol. Biol., vol. 235, No. 1447-1558, 1994.*
Yong et al. Chem. Res. Toxicol. vol. 9, pp. 179-187, 1996.*
Hacker et al. Biochem. vol. 36, pp. 14,080-14,087, 1997.*
Raney et al. Proc. Natl. Acad. Sci., USA, vol. 91, pp. 6644-6648, 1994.
Dennis Regina
Morris Patrick
Raney Kevin
Adler Benjamin Aaron
Board of Trustees of the University of Arkansas
Wang Andrew
Zara Jane
LandOfFree
Screening methods for compounds that inhibit or stimulate... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Screening methods for compounds that inhibit or stimulate..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Screening methods for compounds that inhibit or stimulate... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2616239