The present invention relates to the recombinant DNA which encodes the N.BstNBI nicking endonuclease and modification methylase, and the production of N.BstNBI nicking endonuclease from the recombinant DNA. N.BstNBI nicking endonuclease is originally isolated from Bacillus stearothermophilus. It recognizes a simple asymmetric sequence, 5' GAGTC 3', and it cleaves only one DNA strand, 4 bases away from the 3'-end of its recognition site.
Restriction endonucleases are enzymes that recognize and cleave specific DNA sequences. Usually there is a corresponding DNA methyltransferase that methylates and therefore protects the endogenous host DNA from the digestion of a certain restriction endonuclease. Restriction endonucleases can be classified into three groups: type I, II, and III. More than 3000 restriction endonucleases with over two hundred different specificities have been isolated from bacteria (Roberts and Macelis, Nucleic Acids Res. 26:338-350 (1998)). Type II and type IIs restriction enzymes cleave DNA at a specific position, and therefore are useful in genetic engineering and molecular cloning.
Most restriction endonucleases catalyze double-stranded cleavage on DNA substrate via hydrolysis of two phosphodiester bonds on two DNA strands (Heitman, Genetic Engineering 15:57-107 (1993)). For example, type II enzymes, such as EcoRI and EcoRV, recognize palindromic sequences and cleave both strands symmetrically within the recognition sequence. Type IIs endonucleases recognize asymmetric DNA sequences and cleave both DNA strands outside of the recognition sequence.
There are some proteins in the literature which break only one DNA strand and therefore introduce a nick into the DNA molecule. Most of those proteins are involved in DNA replication, DNA repair, and other DNA-related metabolisms (Kornberg and Baker, DNA replication. 2nd edit. W. H. Freeman and Company, New York, (1992)). For example, gpII protein of bacteriophage fI recognizes and binds a very complicated sequence at the replication origin. It introduces a nick in the plus strand, which initiates rolling circle replication, and it is also involved in circularizing the plus strand to generate single-stranded circular phage DNA. (Geider et al., J. Biol. Chem. 257:6488-6493 (1982); Higashitani et al., J. Mol. Biol. 237:388-400 (1994)). Another example is the MutH protein, which is involved in DNA mismatch repair in E. coli. MutH binds at dam methylation site (GATC), where it forms a protein complex with nearby MutS which binds to a mismatch. The MutL protein facilitates this interaction and this triggers single-stranded cleavage by MutH at the 5' end of the unmethylated GATC site. The nick is then translated by an exonuclease to remove the mismatched nucleotide (Modrich, J. Biol. Chem. 264:6597-6600 (1989)).
The nicking enzymes mentioned above are not very useful in the laboratory for manipulating DNA due to the fact that they usually recognize long, complicated sequences and usually associate with other proteins to form protein complexes which are difficult to manufacture. Thus none of these nicking proteins are commercially available. Recently, we have found a nicking protein, N.BstNBI, from the thermophilic bacterium Bacillus stearothermophilus, which is an isoschizomer of N.BstSEI (Abdurashitov et al., Mol. Biol. (Mosk) 30:1261-1267 (1996)). Unlike gpII and MutH, N.BstNBI behaves like a restriction endonuclease. It recognizes a simple asymmetric sequence, 5' GAGTC 3', and it cleaves only one DNA strand, 4 bases away from the 3'-end of its recognition site (FIG. 1A).
Because N.BstNBI acts more like a restriction endonuclease, it should be useful in DNA engineering. For example, it can be used to generate a DNA substrate containing a nick at a specific position. N.BstNBI can also be used to generate DNA with gaps, long overhangs, or other structures. DNA templates containing a nick or gap are useful substrate for researchers in studying DNA replication, DNA repair and other DNA related subjects (Kornberg and Baker, DNA replication. 2nd edit. W. H. Freeman and Company, New York, (1992)). A potential application of the nicking endonuclease is its use in strand displacement amplification (SDA), which is an isothermal DNA amplification technology. SDA provides an alternative to polymerase chain reaction (PCR), and it can reach 10.sup.6 -fold amplification in 30 minutes without thermo-cycling (Walker et al., Proc. Natl. Acad. Sci. USA 89:392-396 (1992)). SDA uses a restriction enzyme to nick the DNA and a DNA polymerase to extend the 3'-OH end of the nick and displace the downstream DNA strand (Walker et al., (1992)). The SDA assay provides a simple (no temperature cycling, only incubation at 60.degree. C.) and very rapid (as short as 15 minutes) detection method and can be used to detect viral or bacterial DNA. SDA is being introduced as a diagnostic method to detect infectious agents, such as Mycobacterium tuberculosis and Chlamydia trachomatis (Walker and Linn, Clin. Chem. 42:1604-1608 (1996); Spears et al., Anal. Biochem. 247:130-137 (1997)).
For SDA to work, a nick has to be introduced into the DNA template by a restriction enzyme. Most restriction endonucleases make double-stranded cleavages. Therefore, modified .alpha.-thio deoxynucleotides (dNTP.alpha.S) have to be incorporated into the DNA, so that the endonuclease only cleaves the unmodified strand which is within the primer region (Walker et al., 1992). The a-thio deoxynucleotides are eight times more expensive than regular dNTPs (Pharmacia), and is not incorporated well by the Bst DNA polymerase as compared to regular deoxynucleotides (J. Aliotta, L. Higgins, and H. Kong, unpublished observation).
Alternatively, if a nicking endonuclease is used in SDA, it will introduce a nick into the DNA template naturally. Thus the dNTP.alpha.S is no longer needed for the SDA reaction when a nicking endonuclease is being used. This idea has been tested, and the result agreed with our speculation. The target DNA can be amplified in the presence of the nicking endonuclease N.BstNBI, dNTPs, and Bst DNA polymerase.
With the advent of genetic engineering technology, it is now possible to clone genes and to produce the proteins that they encode in greater quantities than are obtainable by conventional purification techniques. Type II restriction-modification systems are being cloned with increasing frequency. The first cloned systems used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Molec. Gen. Genet 178:717-719 (1980); HhaII: Mann et al., Gene 3:97-112 (1978); PstI: Walder et al., Proc. Nat. Acad. Sci. 78:1503-1507 (1981)). Since the presence of restriction-modification systems in bacteria enable them to resist infection by bacteriophages, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from libraries that have been exposed to phage. This method has been found, however, to have only limited value. Specifically, it has been found that cloned restriction-modification genes do not always manifest sufficient phage resistance to confer selective survival.
Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning plasmids (EcoRV: Bougueleret et al., Nucl. Acids Res. 12:3659-3676 (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80:402-406 (1983); Theriault and Roy, Gene 19:355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol. 164:501-509 (1985)).
A further approach which is being used to clone a growing number of systems involves selection for an active methylase gene (refer to U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al., Nucl. Acids Res. 13:6403-6421 (1985)). Since restriction and modification genes are often closely linked, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but instead yields only the methylase gene (BspRI: Szomolanyi et al., Gene 10:219-225 (1980); BcnI: Janulaitis et al, Gene 20:197-204 (1982); BsuRI: Kiss and Baldauf, Gene 21:111-119 (1983); and MspI: Walder et al., J. Biol. Chem. 258:1235-1241 (1983)).
Another method for cloning methylase and endonuclease genes is based on a colorimetric assay for DNA damage (see U.S. Pat. No. 5,492,823). When screening for a methylase, the plasmid library is transformed into the host E.coli strain such as AP1-200. The expression of a methylase will induce the SOS response in an E.coli strain which is McrA+, McrBC+, or Mrr+. The AP1-200 strain is temperature sensitive for the Mcr and Mrr systems and includes a lac-Z gene fused to the damage inducible dinD locus of E.coli. The detection of recombinant plasmids encoding a methylase or endonuclease gene is based on induction at the restrictive temperature of the lacZ gene. Transformants encoding methylase genes are detected on LB agar plates containing X-gal as blue colonies. (Piekarowicz et al., Nucleic Acids Res. 19:1831-1835 (1991) and Piekarowicz et al., J. Bacteriology 173:150-155 (1991)). Likewise, the E.coli strain ER1992 contains a dinD1-Lac Z fusion but is lacking the methylation dependent restriction systems McrA, McrBC and Mrr. In this system (called the "endo-blue" method), the endonuclease gene can be detected in the absence of it's cognate methylase when the endonuclease damages the host cell DNA, inducing the SOS response. The SOS-induced cells form deep blue colonies on LB agar plates supplemented with X-gal. (Fomenkov et al., Nucleic Acids Res. 22:2399-2403 (1994))
Sometimes the straight-forward methylase selection method fails to yield a methylase (and/or endonuclease) clone due to various obstacles, See, e.g., Lunnen et al., Gene 74(1):25-32 (1988). One potential obstacle to cloning restriction-modification genes lies in trying to introduce the endonuclease gene into a host not already protected by modification. If the methylase gene and endonuclease gene are introduced together as a single clone, the methylase must protectively modify the host DNA before the endonuclease has the opportunity to cleave it. On occasion, therefore, it might only be possible to clone the genes sequentially, methylase first then endonuclease (see U.S. Pat. No. 5,320,957).
Another obstacle to cloning restriction-modification systems lies in the discovery that some strains of E.coli react adversely to cytosine or adenine modification; they possess systems that destroy DNA containing methylated cytosine (Raleigh and Wilson, Proc. Natl. Acad. Sci. USA 83:9070-9074 (1986)) or methylated adenine (Heitman and Model, J. Bacteriology 196:3243-3250 (1987); Raleigh et al., Genetics 122:279-296 (1989); Waite-Rees et al., J. Bacteriology 173:5207-5219 (1991)). Cytosine-specific or adenine-specific methylase genes cannot be cloned easily into these strains, either on their own, or together with their corresponding endonuclease genes. To avoid this problem it is necessary to use mutant strains of E. coli (McrA- and McrB- or Mrr-) in which these systems are defective.
An additional potential difficulty is that some restriction endonuclease and methylase genes may not express in E. coli due to differences in the transcription machinery of the source organism and E. coli, such as differences in promoter and ribosome binding sites. The methylase selection technique requires that the methylase express well enough in E. coli to fully protect at least some of the plasmids carrying the gene.
Because purified restriction endonucleases, and to a lesser extent modification methylases, are useful tools for characterizing genes in the laboratory, there is a commercial incentive to obtain bacterial strains through recombinant DNA techniques that synthesize these enzymes in abundance. Such strains would be useful because they would simplify the task of purification as well as provide the means for production in commercially useful amounts.