Deoxyribonuclease

Details Deoxyribonuclease Deoxyribonuclease

1999-11-24

2001-08-21

Patterson, Jr., Charles L. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

Reexamination Certificate

active

06277614

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a useful deoxyribonuclease as a reagent for gene manipulation and to a process for producing said enzyme using genetic engineering techniques.
2. Description of the Related Art
The enzymes most frequently used as deoxyribonucleases that recognize specific sequences and cleave double-stranded DNAs (that is, genes), base sequence-specifically, are those called restriction enzymes, and they typically recognize the sequences of 4 to 8 bases. Restriction enzymes have played essential roles in genetic engineering experiments: they have been used in daily gene manipulation experiments, and thereby greatly contributed to advances in molecular medicine, molecular biology, and biochemistry.
In addition to restriction enzymes, as enzymes that recognize nucleotide sequence and cleave double-stranded DNAs, there is a group of enzymes called homing endonucleases, which are involved in the DNA recombination process. Although these enzymes generally require sequences as long as 20 or more bases for recognition, recognized sequences are specific for respective enzymes, and therefore, these enzymes can be used for the purpose of site-specific DNA cleavage.
Thus, there have been many practical applications of enzymes that recognize and cleave DNA sequences. As to enzymes that recognize and cleave specific steric structures of DNA, however, only few studies are known with particular emphasis on
Escherichia coli
RuvC protein, and there are no enzymes that have already come into practical use, although biochemical properties such as substrate specificity have been elucidated to some extent.
Although several enzymes that recognize and cleave a specific steric structure such as the Holliday structured are presently known, they are all derived from mesophilic organisms, and their thermostability or cleavage efficiency in vitro is low. An object of the present invention is to develop a practical enzyme that specifically recognize and cleave a Holliday structured DNA, which is a DNA recombination intermediate, to resolve it into two sets of double-stranded DNAs, and to provide such enzyme as a reagent for gene manipulation.
SUMMARY OF THE INVENTION
The present inventors have discovered, as a result of their extensive study, a novel protein having deoxyribonuclease activity from a hyperthermophilic archaebacterium,
Pyrococcus furiosus
, and succeeded in cloning the gene encoding said protein. Furthermore, the present inventors have succeeded in preparing a transformant into which the gene has been introduced, and in producing said protein on a large scale.
Thus, the present invention relates to a protein which is thermostable and which has deoxyribonuclease activity that specifically acts on and cleaves a Holliday structured DNA, which is an intermediate structure in the DNA recombination process, to resolve it into two sets of double-stranded DNAs.
The present invention also relates to a gene encoding said protein.
The present invention further relates to a process for producing said protein, comprising:
1) preparing said gene,
2) constructing an expression vector by inserting said gene into a vector,
3) transforming host cells with said vector,
4) culturing said transformants, and
5) isolating said protein from said culture.
The present inventors have screened archaebacteria for a protein having activity that specifically cleaves an artificially synthesized Holliday structured DNA to resolve it into two sets of double-stranded DNAs, with the aim of isolating a resolvase that specifically recognize and cleave a Holliday structured (cruciform structure) DNA, which is an intermediate in the DNA recombination process. As a result, the present inventors have discovered a protein having desired properties from
Pyrococcus furiosus
, a species of hyperthermophilic archaebacteria.
The gene region encoding the protein was cloned, and determination of the base sequence of the gene obtained revealed a small open reading frame (ORF) consisting of 123 amino acids. Only the ORF was then subcloned, and the protein was produced in
E. coli
. The protein obtained was purified until an electrophoretically single band was obtained, and its substrate specificity was studied in detail. In result, it was found that the enzyme has the desired activity that specifically recognizes and cleaves only four-way junction Holliday structured as shown in
FIG. 1
or
2
. The other structures such as single-stranded, double-stranded, loop-out structures, a single base mismatch, and the like were not cleaved, although, in some cases, a quite small amount of cleavage products was observed with three-way junction structures. When a large excess (10-fold equivalents) of the enzyme was added to the reaction, the cleavage efficiency for the three-way junction structures was enhanced, and also the loop-out structure was slightly cleaved.
Regarding the cleavage pattern of the Holliday structured, it was confirmed, based on the re-ligation of cleaved DNA strands by DNA ligase reaction, that a phosphodiester bond is cleaved into 5′-phosphate and 3′-OH forms.
Based on the above results, it was concluded that the protein is a novel, DNA steric structure-specific deoxyribonuclease, and the protein was named “Pfu-HJC endonuclease”. Database search regarding amino acid sequence revealed that no proteins having known functions that exhibit a significant homology (or genes encoding the same) have been registered. However, an ORF having a highly homologous sequence was found in all the four archaebacteria of which entire genomic sequences have been decoded to date (
Methanococcus jannaschii, Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum
, and
Pyrococcus horikoshii
), strongly suggesting that the enzyme may be an important enzyme that is conserved at least among archaebacteria.


REFERENCES:
Komori, et al. (1999) Proc Natl. Acas. Sci., USA 96, 8873-8878.*
“Recognition and Manipulation of Branched DNA Structure by Junction-resolving Enzynes” (J. Mol. Biol (1977) 269, pp. 647-664) (White, et al.).
“Holliday Junction Processing in Bacteria: Insights from Evolutionary Conservation in RuvABC, RecG, and RusA” (Journal of Bacteriology, Sep. 1999, pp. 5543-5550) (Sharpley, et al.).
“Processing the Holliday junction in homologous recombination ”(TIBS 21 Mar. 1996, pp. 107-111) (Shinagawa, et al.).
Kayoko Komori et al., “PI-Pful and PI-Pfull, intein-induced homing endonucleases fromPyrococcus furiosus. I. Purification and identification of the homing-type endonuclease activities”,Nucleic Acids Research, vol. 27, No. 21, pp. 4167-4174 (1999).
Joan Riera et al., “Ribonucleotide reductase in the archaeonPyrococcus furiosus: A critical enzyme in the evolution of DNA genomes?”,Proc. Natl. Acad. Sci. USA, vol. 94, pp. 475-478 (Jan. 1997).
Steven J. Sandler et al., “recA-Like genes from three archaen species with putative protein products similar to Rad51 and Dmc1 proteins of the yeastSaccharomyces cerevisiae”,Nucleic Acids Research, vol. 25, No. 11, pp. 2125-2132 (1996).
Norimichi Nomura et al., “Molecular Characterization and Postsplicing Fate of Three Introns within the Single rRNA Operon of the Hyperthermophilic ArchaeonAeropyrum pernixK1”,Journal of Bacteriology, vol. 180, No. 14, pp. 3635-3643, (Jul. 1998).
Hiromi Daiyasu et al., “Hjc resolvase is a distantly related member of the type II restriction endonuclease family”,Nucleic Acids Research, vol. 28, No. 22, pp. 4540-4543 (2000).

Affiliated with

Also associated with

Rating

Deoxyribonuclease does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Deoxyribonuclease, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Deoxyribonuclease will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2436131