Endonuclease compositions and methods of use

Details Endonuclease compositions and methods of use Endonuclease compositions and methods of use

1998-12-11

2002-09-24

Priebe, Scott D. (Department: 1632)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S069100, C435S320100, C435S325000, C435S455000, C435S456000, C424S093200, C424S093210, C536S023200, C536S023500

Reexamination Certificate

active

06455250

ABSTRACT:

1.0 BACKGROUND OF THE INVENTION
1.1 Field of the Invention
The present invention relates generally to the fields of immunology and oncology. Disclosed are compositions comprising nucleotide sequences encoding endonuclease SR, and polypeptides encoded thereby. Also disclosed are methods for repairing DNA and modulating genetic recombination in a cell, particularly through the use of a specific endonuclease, which is referred to hereinafter as Endo-SR (
Endo
nuclease implicated in
S
witch
R
ecombination). The compositions disclosed are useful in cleaving DNA at specific G-rich regions which are implicated in modulating DNA rearrangements. Also disclosed are methods for the design and isolation of peptidomimetics and Endo-SR inhibitors useful in the treatment of leukemias, lymphomas, and other cancers, as well as modulation of apoptosis and programmed cell death events.
1.2 Description of Related Art
1.2.1 B-Lymphocytes
Mature B lymphocytes can alter their Ig isotype expression by targeted deletional rearrangement of their IgH constant region genes in a process called isotype switch recombination (Harriman et al., 1993; Shimizu and Honjo, 1984). The ability of B cells to “switch” one Ig constant region for another significantly enhances the versatility of the immune system by allowing activated B cells to selectively alter the function of their secreted Ig without altering their ligand specificity (Esser and Radbruch, 1990; Mond et al., 1995a,b).
Recombination breakpoints generated by this process primarily map to large (approximately 1 to 10 kb), highly-repetitive DNA regions found upstream of all IgH constant region genes (Esser and Radbruch, 1990; Harriman et al., 1993). These switch regions are primarily composed of degenerate, variable-length G-rich repeat units, that differ considerably both within and between species (Esser and Radbruch, 1990; Harriman et al., 1993). However, all switch regions contain disproportionate numbers of two pentamer motifs, TGGGN and TGAGC, that are found at or adjacent to most analyzed switch recombination breakpoints (Dunnick et al., 1993; Mowatt et al., 1986; Kenter et al., 1993, Petrini and Dunnick, 1989; Wuerffel et al., 1992).
Selective recombination of individual switch regions directly correlates with transcriptional activation of these regions in response to extracellular signals (Berton and Vitetta, 1990; Bottaro et al., 1994; Kuwabara et al., 1995; Rothman et al., 1990; Warren and Berton, 1995; Xu et al., 1993; Xu and Stavnezer, 1992). Recent studies have demonstrated that an additional signal(s) is required to induce switch recombination at an actively transcribed switch region (Bottaro et al., 1994). Specific transcriptional induction has thus been proposed to increase the accessibility of a particular switch region to switch recombinase factors, which may also be activated by some of the same signals responsible for transcriptional activation of these loci (Mandler et al, 1993; Purkerson and Isakson, 1992; Snapper and Mond, 1993).
The search for switch recombinase factors has primarily focused on the detection of DNA binding factors which specifically interact with switch repeat sequences, and although this approach has lead to the characterization of several novel DNA binding factors (Fukita et al., 1993; Marcu et al., 1992; Miranda et al., 1995; Mizuta et al., 1993; Waters et al., 1989; Wuerffel et al., 1990; Xu et al., 1992), as yet none of these factors have been directly implicated in the switch recombination reaction.
2.0 SUMMARY OF THE INVENTION
The present invention overcomes limitations in the prior art by providing novel endonuclease polypeptides, and in particular, mammalian Endo-SR polypeptides which have specific endonuclease activity. In a preferred embodiment, the Endo-SR polypeptide is isolated from mammalian sources, including murine, bovine, and human sources, or from organisms such as
C. elegans
and the like, and comprises at least a 19-contiguous amino acid sequence from SEQ ID NO:2. More preferably, the polypeptide comprises the amino acid sequence of SEQ ID NO:2, and is encoded by a polynucleotide that comprises at least a 76-contiguous nucleic acid sequence from SEQ ID NO:1, and preferably comprising the sequence of SEQ ID NO: 1. Exemplary murine genomic DNA sequences include sequences such as those in SEQ ID NO:3 and SEQ ID NO:4. Preferred sequences include sequences comprising at least 49 contiguous nucleic acid sequences from SEQ ID NO:3, and sequences comprising at least 24 contiguous nucleic acid sequences from SEQ ID NO:4. Sequences comprising at least 204 contiguous nucleic acids from SEQ ID NO:4 are also contemplated to be particularly useful.
The Endo-SR polypeptides of the present invention preferentially cleave either a “TGGGN” or a “TGAGC” polynucleotide sequence, and more preferably cleave both of these sequences, which are known as “switch pentamer motifs” at or near recombination breakpoints. These polypeptides have been shown to be enriched in lymphoid tissue nuclear extracts.
In one important embodiment, the invention provides an isolated and purified amino acid segment comprising Endo-SR polypeptide comprising the amino acid sequence of SEQ ID NO:2. This polypeptide is a murine polypeptide, the coding region for which is given in SEQ ID NO:1 (a cDNA clone). The corresponding murine genomic DNA sequences are given in SEQ ID NO:3 and SEQ ID NO:4. The Endo-SR polypeptide exhibits specific endonuclease activity for G-rich DNA sequences. In related embodiments, methods for making and using this protein, derivatives and mutants thereof, and antibodies directed against these proteins are also disclosed. Also disclosed are methods for the design of inhibitors, such as peptidomimetics, of Endo-SR, and their use in modulation of Endo-SR activity.
In another important embodiment, the invention provides an isolated and purified nucleic acid segment comprising the mammalian gene which encodes the Endo-SR polypeptide disclosed herein. The nucleotide sequence of the cDNA is given in SEQ ID NO:1, and the partial murine genomic sequences are identified in SEQ ID NO:3 and SEQ ID NO:4. In related embodiments, methods for making, using, altering, mutagenizing, assaying, and quantitating these nucleic acid segments are also disclosed. Also disclosed are diagnostic methods and assay kits for the identification and detection of related gene sequences in a variety of in vitro and in vivo methodologies. Because Endo-SR has been implicated in the process of antibody isotype switch recombination and programmed cell death, highly purified or recombinant Endo-SR represents a useful tool for dissection of the mechanism of switch recombination and/or apoptosis.
Another aspect of the present invention is a mammalian cell, and in particular, a bovine or human cell that produces the novel Endo-SR polypeptide disclosed herein. Exemplary mammalian cells that produce the polypeptide include human HeLa and 293T kidney cells as well as mouse L-cell and NIH3T3.
A further aspect of the present invention is a vector, such as a plasmid, cosmid, phage, virus, phagemid, bacterial artificial chromosome or yeast artificial chromosome, that contains a nucleic acid sequence comprising a whole or a portion of a gene encoding Endo-SR (cDNA, SEQ NO ID:1; genomic clones, SEQ ID NO:3 and SEQ ID NO:4). Also provided is a transformed host cell comprising a native or recombinant gene encoding Endo-SR, as well as a tissue culture, a cell culture, or an animal, fungal or bacterial cell culture or suspension or lysate of a host cell transformed with such a vector.
The invention also provides a pharmaceutical composition comprising an Endo-SR polypeptide or a gene encoding such a polypeptide.
Because moderate levels of enzymatically active Endo-SR have been detected in animal serum and the Endo-SR is secreted, the polypeptide may play a role in preventing DNA accumulation in blood and tissues. Monoclonal or polyclonal antibodies directed towards the Endo-SR protein and/or Endo-SR peptides may prove to have therapeutic potential for patients carryi

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