Human gene encoding 3′-5′ exonuclease

Details Human gene encoding 3′-5′ exonuclease Human gene encoding 3′-5′ exonuclease

2000-08-08

2003-10-14

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

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S252300, C435S199000, C536S023200

Reexamination Certificate

active

06632665

ABSTRACT:

BACKGROUND OF THE INVENTION
There are a growing number of antineoplastic and antiviral agents such as the nucleoside analogs and dideoxy nucleosides that act as anti-metabolites by inhibiting nucleic acid polymerization, or elongation. Some resistance or ineffectiveness of these agents may be due to an exonuclease activity that removes the analog from the nucleic acid molecule and permits the analog to be replaced with the correct nucleoside.
As an example of such a therapy for treatment of acute myeloblastic leukemia (AML) includes administration of 1-&bgr;-D-arabinofuranosylcytosine (araC), an analog of dCTP and potent inhibitor of DNA replication. For a review, see Gilman, et al. (Eds.),
The Pharmacological Basis of Therapeutics
, Eighth Edition, Pergamon Press; New York (1990), pp. 1230-1232. Despite the well established therapeutic value of araC, the precise mechanism by which cell death is induced is unclear. One possibility is that inhibition of DNA synthesis without concomitant suppression of RNA and protein synthesis leads to “unbalanced growth” resulting in increased cell volume and ultimately cell death. In araC treatment, it has been observed that a large number of AML patients are initially refractory to the drug or later develop resistance to araC resulting in failure of therapy in the long term. It is believed that araC resistance arises in part from the relative activities of metabolic enzymes that participate in conversion of araC to araCTP and ultimately to an inactive araUMP. Other factors which may influence araC efficacy include (i) the ability of cells to transport araC, (ii) deoxycytidine kinase deficiency, (iii) increased CTP synthase activity which gives rise to increased intracellular dCTP that may inhibit araC activity, (iv) cytidine deaminase activity, and/or (v) coordinated polymerase/exonuclease activities. Changes in araC structure and/or intracellular concentration relative to analogous compounds may alter affinity of DNA polymerases for araC, thereby resulting in decreased incorporation of the analog into replicating DNA and decreased efficacy of araC chemotherapy regimens.
Thus there exists a need in the art to identify metabolic factors which modulate the ability of chemotherapeutic agents to effect cell killing. Isolation of polypeptides, and their underlying polynucleotide sequences that modulate araC activity would permit the design and identification of therapeutics that regulate the biological activity of the polypeptides and increase efficiency of chemotherapeutic agent at lower doses. Treatment regimens including lower doses of a chemotherapeutic agent may be more easily tolerated in patients, reduce unpleasant side effects, and increase overall efficiency of the treatment program.
SUMMARY OF THE INVENTION
The present invention addresses certain shortcomings in the fields of anti-cancer and anti-viral therapies by providing isolated 3′-5′ exonucleases that are not linked to any polymerase activity, and that are shown herein to be involved in decreasing the effectiveness of certain therapeutic compounds, and in particular by providing an isolated human genomic 3′-5′ exonuclease encoding polynucleotide. For example, agents such as nucleoside analogs and chain-terminating dideoxynucleotides, which are used as therapeutic agents against proliferating cells, are removed from a cellular or viral genome by the disclosed exonucleases during treatment, allowing the cell or virus to continue to proliferate. In light of the present disclosure, these isolated exonucleases may be inhibited or even eliminated from a cell containing an anti-proliferative therapeutic agent in order to increase the effectiveness of such an agent.
Disclosed herein are isolated nucleic acid molecules of from about 708 to about 1642 nucleotides in length that include a gene, or the full length complement of a gene, particularly genes that encode a polypeptide, or protein, that includes the amino acid sequence of those sequences designated herein as SEQ ID NO:2, SEQ ID NO:4, SEQ ID 30, SEQ ID NO:32 or SEQ ID NO:34 and conservative variants of these polypeptides. Conservative variants of a polypeptide typically contain an alternative amino acid at one or more sites within the protein. Substitutions preferably are conservative, that is, one amino acid is replaced with one of similar size and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isolcucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Conservative variants may also include small deletions or insertions of amino acids, so long as the protein maintains its enzymatic activity.
For example, insertional variants may include fusion proteins such as those used to allow rapid purification of the polypeptide and also may include hybrid proteins containing sequences from other proteins and polypeptides such as homologues of the polypeptide. For example, an insertional variant may include portions of the amino acid sequence of the polypeptide from one species, together with portions of the homologous polypeptide from another species. Other insertional variants may include those in which additional amino acids are introduced within the coding sequence of the polypeptide. These typically are smaller insertions than the fusion proteins described above and are introduced, for example, to disrupt a protease cleavage site, or to aid in chromatographic purification of the polypeptide.
Also disclosed are several regions, hereinafter conserved regions, within these amino acid sequences that are evolutionarily conserved between the human, murine and Drosophila polypeptides or proteins. For example, in one particular region of the polypeptides disclosed herein that includes a contiguous sequence from about amino acids 12 through 25 of SEQ ID NO:2, about amino acids 8 through 21 of SEQ ID NO:4, about amino acids 12 through 25 of SEQ ID NO:30, about amino acids 8 through 21 of SEQ ID NO:32 and about amino acids 18 through 31 of SEQ ID NO:34 is substantially conserved between these three species and may be so among other species as well. Additionally, a second region of conserved amino acid sequence is disclosed herein to be from about amino acid 124 through about 134 of SEQ ID NO:2, from about amino acid 113 or about 117 through about 127 of SEQ ID NO:4, from about amino acid 120 or 124 through about 134 of SEQ ID NO:30, from about amino acid 117 through about 127 of SEQ ID NO:32 and from about amino acid 129 or 133 through about 143 of SEQ ID NO:34 is substantially conserved between these three species and may be so among others. Furthermore, a third region from about amino acids 195 through 205 of SEQ ID NO:2, 188 through 198 of SEQ ID NO:4, 195 through 205 of SEQ ID NO:30, 188 through 198 of SEQ ID NO:32 and 303 through 313 of SEQ ID NO:34 is also conserved among the three species and may be conserved among other species. The disclosed invention also encompasses mutations of the conserved regions which may be conservative in nature, or may be targeted to disrupt or modify enzymatic activity of the polypeptides or may be targeted to disrupt or modify potential interactions with other molecules.
In certain embodiments, the polypeptides or proteins disclosed herein are encoded by the nucleic acid sequences designated herein as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33 or the complement, or full length complement of any of these. As used herein the term “complement” is used to define a second strand of nucleic acid that will hybridi

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