Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for...
Reexamination Certificate
2001-06-01
2002-05-28
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
C435S069100
Reexamination Certificate
active
06395523
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to methods for converting Type IIs restriction endonucleases into site specific nicking endonucleases. The engineering theme is based on a naturally existing nicking endonuclease, N.BstNBI, which is related to Type IIs restriction endonucleases. In general, Type IIs endonucleases bind to a specific sequence and cleave both DNA strands near, but not within the specific sequence. The double-stranded cleavage activity of N.BstNBI has been severely limited by natural mutations and thus it nicks only one strand of DNA under standard digestion conditions. In accordance with the present invention, new nicking endonucleases can be engineered from Type IIs endonucleases by either inactivating their second-strand cleavage activity or by swapping the cleavage domains between a target Type IIs enzyme and a known or engineered nicking enzyme.
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 digestion by its cognate restriction endonuclease. Restriction endonucleases can be classified into three groups based on cofactor requirements: Type I, II (including IIs), 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 require only Mg
++
as cofactor; both cleave DNA at a specific position, and therefore are useful in genetic engineering and molecular cloning.
Most restriction endonucleases catalyze double-stranded cleavage of DNA substrate via hydrolysis of two phosphodiester bonds on opposite 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 events (Komberg 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 of the phage genome. It introduces a nick in the plus strand to initiate rolling circle replication; it is also involved in ligating the displaced 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, triggering 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/or are associated with other proteins to form protein complexes which are difficult to manufacture and use. None of these nicking proteins are commercially available. The nicking enzyme N.BstNBI, was found from the thermophilic bacterium
Bacillus stearothermophilus
(Morgan et al.,
Biol. Chem
. 381:1123-1125 (2000); U.S. Pat. No. 6,191,267). N.BstNBI 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, without interaction with other proteins.
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 substrates 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)). One 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). It can reach 10
6
-fold amplification in 30 minutes without thermo-cycling. 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.,
Proc. Natl. Acad. Sci
. USA. 89:392-396 (1992)). The SDA assay provides a simple (no temperature cycling, only incubation at 60° 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, in previous work, substituted &agr;-thio deoxynucleotides (dNTP&agr;S) have been incorporated into the DNA. Many restriction endonucleases will not cleave phosphodiester bonds with (&agr;-thio substitutions. Thus the endonuclease only cleaves the un-substituted linkages which are designed to be within the primer region. The (&agr;-thio deoxynucleotides are eight times more expensive than regular dNTPs (Pharmacia), and are 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 were to be used in SDA, it would introduce a nick into the DNA template naturally. Thus the dNTP&agr;S would no longer be 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 (U.S. Pat. No. 6,191,267).
There is an increasing demand for more nicking endonucleases, because they are useful in SDA and other DNA engineering applications. We have cloned and characterized the nicking endonuclease N.BstNBI and our results show that N.BstNBI is a naturally mutated Type IIs endonuclease with diminished double-stranded cleavage activity (U.S. Pat. No. 6,191,267). The natural occurrence of this type of endonuclease may be quite limited; in any event, assay methods to detect them unambiguously are not available. So far only two nicking endonucleases have been reported and both recognize same specificity (U.S. Pat. No. 6,191,267). The methods disclosed herein provide a novel approach for generating new nicking endonucleases using a protein engineering approach.
Effort has been long taken to engineer novel endonucleases with little success. FokI is a Type IIs restriction enzyme which exhibits a bipartite nature, an N-terminal DNA recognition domain and a C-terminal DNA cleavage domain (Wah et al., Nature 388:97-100 (1997)). The modular nature of FokI led to the invention of several enzymes with new specificities by substituting other DNA binding proteins for
Besnier Caroline
Kong Huimin
Xu Yan
Horlick Kenneth R.
New England Biolabs Inc.
Strzelecka Teresa
Williams Gregory D.
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