Nucleic acid enzymes for cleaving DNA

Details Nucleic acid enzymes for cleaving DNA Nucleic acid enzymes for cleaving DNA

1994-01-19

2001-02-27

Schwartzman, Robert A. (Department: 1635)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120, C435S325000, C435S333000, C536S023100, C536S023200, C536S024500

Reexamination Certificate

active

06194180

ABSTRACT:

TECHNICAL FIELD
The present invention relates to nucleic acid enzymes for cleaving DNA.
BACKGROUND
Some genes have their coding sequences interrupted by stretches of non-coding DNA. These non-coding sequences are termed introns. To produce a mature transcript from these genes, the primary RNA transcript (precursor RNA) must undergo a cleavage-ligation reaction termed RNA splicing. This RNA splicing produces the mature transcript of the polypeptide coding messenger RNA (mRNA), ribosomal RNA, or transfer RNA (tRNA). Introns are grouped into four categories (Groups I, II, III, and IV) based on their structure and the type of splicing reaction they undergo.
Of particular interest to the present invention are the Group I introns. Group I introns undergo an intra-molecular RNA splicing reaction leading to cyclization that does not require protein cofactors, Cech,
Science,
236:1532-1539 (1987).
The Group I introns, including the intron isolated from the large ribosomal RNA precursor of
Tetrahymena thermophila
, have been shown to catalyze a sequence-specific phosphoester transfer reaction involving RNA substrates. Zaug and Cech, Science, 229:1060-1064 (1985); and Kay and Inoue, Nature, 327:343-346 (1987). This sequence-specific phosphoester transfer reaction leads to the removal of the Group I intron from the precursor RNA and ligation of two exons in a process known as RNA splicing. Splicing reaction catalyzed by Group I introns proceeds via a two-step transesterification mechanism. The details of this reaction have been recently reviewed by Cech,
Science,
236:1532-1539 (1987).
The splicing reaction of Group I introns is initiated by the binding of guanosine or a guanosine nucleotide to a site within the Group I intron structure. Attack at the 5′ splice site by the 3′-hydroxyl group of guanosine results in the covalent linkage of guanosine to the 5′ end of the intervening intron sequence. This reaction generates a new 3′-hydroxyl group on the uridine at the 3′ terminus of the 5′ exon. The 5′ exon subsequently attacks the 3′ splice site, yielding spliced exons and the full-length linear form of the Group I intron.
The linear Group I intron usually cyclizes following splicing. Cyclization occurs via a third transesterification reaction, involving attack of the 3′-terminal guanosine at an interval site near the 5′ end of the intron. The Group I introns also undergo sequence specific hydrolysis reaction at the splice site sequences as described by Inoue et al.,
J. Mol. Biol.,
189:143-165 (1986). This activity has been used to cleave RNA substrates in a sequence specific manner by Zaug et al.,
Nature,
324:429-433 (1986).
The structure of Group I introns has been recently reviewed by J. Burke,
Gene,
73:273-294 (1988). The structure is characterized by nine base paired regions, termed P1-P9 as described in Burke et al.,
Nucleic Acids Res.,
15:7217-7221 (1987). The folded structure of the intron is clearly important for the catalytic activity of the Group I introns as evidenced by the loss of catalytic activity under conditions where the intron is denatured. In addition, mutations that disrupt essential base-paired regions of the Group I introns result in a loss of catalytic activity. Burke,
Gene,
73:273-294 (1988). Compensatory mutations or second-site mutations that restore base-pairing in these regions also restore catalytic activity. Williamson et al.,
J. Biol. Chem.,
262:14672-14682 (1987); and Burke,
Gene,
73:273-294 (1988).
Several different deletions that remove a large nucleotide segment from the Group I introns (
FIG. 2
) without destroying its ability to cleave RNA have been reported. Burke,
Gene,
73:273-294 (1988). However, attempts to combine large deletions have resulted in both active and inactive introns. Joyce et al.,
Nucleic Acid Res.,
17:7879 (1989).
To date, Group I introns have been shown to cleave substrates containing either RNA, or RNA and DNA. Zaug et al.,
Science,
231:470-475 (1986); Sugimoto et al.,
Nucleic Acids Res.,
17:355-371 (1989); and Cech,
Science,
236:1532-1539 (1987). A DNA containing 5 deoxycytosines was shown not to be a cleavage substrate for the Tetrahymena IVS, a Group I intron by Zaug et al.,
Science,
231:470-475 (1986).
BRIEF SUMMARY OF THE INVENTION
It has now been discovered that Group I introns have the ability to cleave single-stranded DNA substrates in a site specific manner.
Therefore the present invention provides a method of cleaving single-stranded DNA at the 3′-terminus of a predetermined nucleotide sequence present within single-stranded DNA. The single-stranded DNA is treated under DNA cleaving conditions with an effective amount of an endodeoxyribonuclease of the present invention where the DNA cleaving conditions include the presence of MgCl
2
at a concentration of at least 20 millimolar.
The present invention also contemplates a composition containing an endodeoxyribonuclease enzyme of the present invention, single-stranded DNA and magnesium ion at a concentration of greater than 20 millimolar.
The present invention further contemplates an endodeoxyribonuclease enzyme capable of cleaving single-stranded DNA at a predetermined nucleotide sequence where the enzyme has a nucleotide sequence defining a recognition site that is contiguous or adjacent to the 5′-terminus of the nucleotide sequence, a first spacer region located 3′-terminal to the recognition site, a P3[5′] region located 3′-terminal to the first spacer region, a second spacer region located 3′-terminal to the P3[5′] region, a first stem loop located 3′-terminal to the second spacer region, a second stem loop located 3′-terminal to the first stem loop, a third spacer region located 3′-terminal to the second stem loop, and a third stem loop located 3′-terminal to the third spacer region, the third stem loop comprising a 5′ stem portion interrupted by a nucleotide sequence defining a P3[3′] region capable of hybridizing to the P3[5′] region.


REFERENCES:
patent: 4987071 (1991-01-01), Cech et al.
patent: 5180818 (1993-01-01), Cech et al.
Cech,Science 236: 1532-1539 (1987).
Burch,Gene 73: 273-294 (1988).
Sugimoto, et al.,Nucl. Acid Res. 17: 355-371 (1989).
Joyce, et al.,Nucl. Acid Res. 17: 7879-7889 (1989).
Doudna, et al.,Nature 339: 519-522 (1989).

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