Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-03-31
2001-07-24
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S193000, C435S069700, C435S091100, C536S023100, C536S023200
Reexamination Certificate
active
06265196
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to hybrid genes which encode hybrid restriction endonucleases. The hybrid restriction endonucleases are designed to specifically recognize DNA at given base sites and to enzymatically cleave the DNA at distant sites.
More specifically, the present invention relates to a method for enzymatically inactivating a target DNA, to a method for detecting conformational change in a nucleic acid and to hybrid molecules comprised of a sequence-specific nucleic acid binding domain joined to a detection domain. 2. Description of the Related Art
Since their discovery nearly 25 years ago (1), Type II restriction enzymes have played a crucial role in the development of the recombinant DNA technology and the field of molecular biology. The Type II restriction (R) endonucleases and modification (M) methylases are relatively simple bacterial enzymes that recognize specific sequences in duplex DNA. While the former cleave DNA, the latter methylate adenine or cytosine residues within the recognition site so as to protect the host-genome against cleavage by the former. So far, over 2500 restriction and modification enzymes have been identified and these are found in widely diverse organisms (2). These enzymes fall into numerous “isoschizomer” (identically cleaving) groups with about 200 sequence-specificities.
Discovery of new enzymes involves tedious and time-consuming effort that requires extensive screening of bacteria and other microorganisms (3). Even when one finds a new enzyme, more often than not, it falls into the already-discovered isoschizomer groups. Furthermore, most naturally occurring restriction enzymes recognize sequences that are 4-6 bp long. Although these enzymes are very useful in manipulating recombinant DNA, they are not suitable for producing large DNA segments. For example, restriction enzymes that recognize DNA sequences 6 bp long, result in cuts as often as every 4096 bases. In many instances, it is preferable to have fewer but longer DNA strands, especially during genome mapping. Rare cutters like NotI, that recognizes 8 bp-long sequences, cut human DNA (which contains about 3 billion bp) every 65536 bases on average. So far, only a few endonucleases with recognition sequences longer than 6 bp (rare cutters) have been identified (New England Biolabs catalog).
R-M (restriction-modification) systems appear, to have a single biological function—namely, to protect cells from infection by foreign DNA that would otherwise destroy them. The phage genomes are usually small. It stands to reason, then, that bacteria select for R-M systems with small recognition sites (4-6 bp) because these sites occur more frequently in the phages. Therefore, a long term goal in the field of restriction-modification enzymes has been to generate restriction endonucleases with longer recognition sites by mutating or engineering existing enzymes (3).
The FokI restriction endonuclease from
Flavobacterium okeanokoites
belongs to the Type IIS class of endonucleases. FokI recognizes the asymmetric sequence 5′-GGATG-3′ and cleaves double-stranded DNA at staggered sites 9 and 13 nucleotides away from the recognition site. The cloning and sequencing of the FokI restriction-modification system have been reported. Several research groups have purified FokI endonuclease and characterized its properties. Previous reports by the present inventor on proteolytic fragments of FokI endonuclease using trypsin have revealed an N-terminal DNA-binding domain and a C-terminal catalytic domain with non-specific DNA cleavage activity (4-7). These reports have suggested that the two domains are connected by a linker region which is susceptible to cleavage by trypsin. The present inventor has also shown that insertion of four (or seven) codons between the recognition and cleavage domains of FokI can alter the cleavage distance of FokI within its substrate.
Recently, Waugh and Sauer have shown that single amino acid substitutions uncouple the DNA-binding and strand scission activities of FokI endonuclease (28). Furthermore, they have obtained a novel class of FokI restriction mutants that cleave hemi-methylated DNA substrates (29). The modular structure of FokI suggested that it may be feasible to construct hybrid endonucleases with novel sequence-specificity by linking other DNA-binding proteins to the cleavage domain of FokI endonuclease. Recently, the present inventor reported the construction of the first “chimeric” restriction endonuclease by linking the Ubx homeo domain to the cleavage domain of FokI (8).
To further probe the linker region, the present inventor constructed several insertion and deletion mutants of FokI endonuclease. A detailed description of the process for making and using and the properties of these mutants are disclosed in U.S. patent application Ser. No. 08/346,293, allowed, the entire contents of which are hereby incorporated by reference and relied upon.
Unlike the Ubx homeo domain, zinc finger proteins, because of their modular structure, offer a better framework for designing chimeric restriction enzymes with tailor-made sequence-specificities. The Cys
2
His
2
zinc finger proteins are a class of DNA-binding proteins that contain sequences of the form (Tyr,Phe)-Xaa-Cys-Xaa
2-4
-Cys-Xaa
3
-Phe-Xaa
5
-Leu-Xaa
2
-His-Xaa
3-5
-His (SEQ ID NOS:1-18) usually in tandem arrays (9). Each of these sequences binds a zinc(ii) ion to form the structural domain termed a zinc finger. These proteins, like many sequence-specific DNA-binding proteins, bind to the DNA by inserting an &agr;-helix into the major groove of the double helix (10).
The crystallographic structure of the three zinc finger domain of zif268 bound to a cognate oligonucleotide reveals that each finger interacts with a triplet within the DNA substrate. Each finger, because of variations of certain key amino acids from one zinc finger to the next, makes its own unique contribution to DNA-binding affinity and specificity.
The zinc fingers, because they appear to bind as independent modules, can be linked together in a peptide designed to bind a predetermined DNA site. Although, more recent studies suggest that the zinc finger—DNA recognition is more complex than originally perceived (11,12), it still appears that zinc finger motifs will provide an excellent framework for designing DNA-binding proteins with a variety of new sequence-specificities.
In theory, one can design a zinc finger for each of the 64 possible triplet codons and, using a combination of these fingers, one could design a protein for sequence-specific recognition of any segment of DNA. Studies to understand the rules relating to zinc finger sequences/DNA-binding preferences and redesigning of DNA-binding specificities of zinc finger proteins are well underway (13-15).
An alternative approach to the design of zinc finger proteins with new specificities involves the selection of desirable mutants from a library of randomized fingers displayed on phage (16-20). The ability to design or select zinc fingers with desired specificity implies that DNA-binding proteins containing zinc fingers will be made to order. Therefore, we reasoned that one could design “artificial” nucleases that will cut DNA at any preferred site by making fusions of zinc finger proteins to the cleavage domain of FokI endonuclease. We thus undertook the deliberate creation of zinc finger hybrid restriction enzymes, the cloning of the hybrid enzymes, and the characterization of their DNA cleavage properties.
One of the main difficulties in cloning or overproducing restriction enzymes is their potential lethality. The restriction enzymes can enzymatically attack and destroy the host DNA. This is circumvented by first cloning a methylase gene (M). The methylase gene modifies the restriction enzyme sites and provides protection against chromosomal cleavage. A restriction endonuclease gene (R) is then introduced into the host on a separate compatible plasmid.
Our work on hybrid restriction endonuclease genes has indicate
Johns Hopkins University
Park Hankyel T.
Pillsbury & Winthrop LLP
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