Patent 6,723,548

The present invention provides polypeptides having pyrimidine glycosylase activity, preferably, pyrimidine glycosylase/AP lyase activity. The polypeptides include a targeting sequence, preferably an exogenous target sequence. The invention includes polynucleotides that include a coding sequence encoding the polypeptides of the present invention. Also provided by the invention are methods of using the polypeptides.

SUMMARY OF THE INVENTION

The present invention represents an advance in the art of repairing DNA lesions that result from, for instance, UV light, oxidative stress, alkylation damage and/or deamination. The introduction to human cells of a glycosylase having the appropriate initiating repair activity would result in cells possessing a fully functional BER pathway. The implications of this would be a faster, more efficient repair of potentially mutagenic and carcinogenic damage. Another benefit would be that this enhanced rate of repair would help to prevent immunosuppression caused by DNA damage. T4-pdg, the glycosylase/AP lyase that can initiate repair at sites of UV induced damage, has been delivered to human cells to increase the repair of damaged DNA; however, the enzyme has not been targeted to the cellular organelles containing the DNA to be repaired, i.e., the nucleus and the mitochondria of a cell. In the present invention, amino acid sequences that promote intracellular nuclear and mitochondrial targeting have been added to enzymes that initiate repair in the BER system.

The present invention provides a polypeptide having pyrimidine glycosylase activity, preferably, pyrimidine glycosylase/AP lyase activity. The polypeptide includes a targeting sequence, preferably an exogenous target sequence. The invention includes a composition that contains the polypeptide and a pharmaceutically acceptable carrier.

In some aspects of the present invention, the polypeptide includes an amino acid sequence of SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43 and a targeting sequence, preferably an exogenous targeting sequence. In other aspects of the present invention, the polypeptide includes an amino acid sequence having pyrimidine glycosylase/AP lyase activity and having at least about 15% identity with an amino acid sequence of SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43, and a targeting sequence, preferably an exogenous targeting sequence.

The present invention is further directed to a polynucleotide that includes a coding sequence encoding a polypeptide having pyrimidine glycosylase activity, preferably pyrimidine glycosylase/AP lyase activity. The polypeptide includes a targeting sequence, preferably, an exogenous targeting sequence.

In some aspects of the present invention, the polynucleotide includes a coding sequence encoding a polypeptide having pyrimidine glycosylase/AP lyase activity and a targeting sequence, preferably, an exogenous coding sequence. The polynucleotide includes a nucleotide sequence of SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:46. In other aspects of the present invention, the polynucleotide includes a coding sequence encoding a polypeptide having pyrimidine glycosylase/AP lyase activity and including a targeting sequence, preferably, an exogenous coding sequence. The polynucleotide includes a nucleotide sequence having at least about 10% identity with a nucleotide sequence of SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:46.

The present invention provides a method for increasing the repair rate of damaged bases in a cell. The method includes introducing to a cell exposed to or at risk of exposure to an agent that damages DNA a composition that includes an amount of a polypeptide effective to increase the repair rate of damaged DNA in the cell compared to a cell that does not include the polypeptide. The polypeptide has pyrimidine glycosylase activity, preferably pyrimidine glycosylase/AP lyase activity, and includes a targeting sequence, preferably, an exogenous targeting sequence.

Also provided is a method for treating mutagenesis in a subject. The method includes introducing to a subject exposed to or at risk of exposure to an agent that damages DNA a composition that includes an effective amount of a polypeptide having pyrimidine glycosylase activity, preferably pyrimidine glycosylase/AP lyase activity, and includes a targeting sequence, preferably, an exogenous targeting sequence.

The present invention provides a method for treating immunosuppression in a subject. The method includes introducing to a subject exposed to or at risk of exposure to an agent that damages DNA a composition that includes an effective amount of a polypeptide having pyrimidine glycosylase activity, preferably pyrimidine glycosylase/AP lyase activity, and includes a targeting sequence, preferably, an exogenous targeting sequence.

Further provided by the present invention is a method for treating tumor formation in a subject. The method includes introducing to a subject exposed to or at risk of exposure to an agent that damages DNA a composition that includes an effective amount of a polypeptide having pyrimidine glycosylase activity, preferably pyrimidine glycosylase/AP lyase activity, and includes a targeting sequence, preferably, an exogenous targeting sequence.

The present invention also provides a method for treating apoptotic cell formation in a subject. The method includes introducing to a subject exposed to or at risk of exposure to an agent that damages DNA a composition that includes an effective amount of a polypeptide having pyrimidine glycosylase activity, preferably pyrimidine glycosylase/AP lyase activity, and includes a targeting sequence, preferably, an exogenous targeting sequence.

Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides polypeptides that have pyrimidine glycosylase activity and a targeting sequence, preferably an exogenous targeting sequence. As used herein, "polypeptide" refers to a polymer of amino acids and does not refer to a specific length of a polymer of amino acids. Thus, for example, the terms peptide, oligopeptide, protein, and enzyme are included within the definition of polypeptide. This term also includes post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. As used herein, "pyrimidine glycosylase" refers to a polypeptide that recognizes the presence of two consecutive damaged bases in a polynucleotide and catalyzes the breakage of the glycosyl bond between the 5' base and the DNA sugar-phosphate backbone. A polypeptide that recognizes the presence of two consecutive damaged pyrimidine bases and catalyzes the breakage of such a bond has "glycosylase activity." Whether a polypeptide has pyrimidine glycosylase activity can be determined by measuring the ability of the polypeptide to cleave the glycosyl bond of the 5' pyrimidine of a cyclobutane pyrimidine dimer in DNA. Such methods are known to the art. A polypeptide having pyrimidine glycosylase activity is often referred to in the art as a pyrimidine dimer-specific DNA glycosylase.

As used herein, "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and single-stranded DNA and RNA. A polynucleotide may include nucleotide sequences having different functions, including, for instance, coding sequences, and non-coding sequences such as regulatory sequences. Coding sequence, non-coding sequence, and regulatory sequence are defined below. A polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. A polynucleotide can be linear or circular in topology. For example, a polynucleotide can be a portion of a vector, such as an expression or cloning vector, or a fragment.

As used herein, "damaged base" and "damaged bases" refers to structural deviations in nucleoside-5'-monophosphates present in a eukaryotic cell's genomic DNA. One type of structural deviation is a covalent joining of the adjacent pyrimidines through the formation of a cyclobutane ring structure at the C5 and C6 positions. Another type of structural deviation is an imidazole ring fragmentation of a purine (either adenine or guanine). The location of such structural deviations in a cell's genomic DNA is referred to as a "lesion." As used herein, "genomic DNA" refers to the DNA present in the nucleus and the mitochondria of a cell. Damaged bases preferably arise from, for instance, UV radiation, ionizing radiation, oxidative stress, alkylation damage, and deamination. Examples of lesions include cis-syn and trans-syn II cyclobutane pyrimidine dimers, FapyA and FapyG (Lloyd, Mutat. Res., 408, 159-170 ((1998), and Lloyd, Progress in Nucleic Acid Research and Molecular Biology, 62, 155-175 (1999)).

Optionally and preferably, a polypeptide of the present invention also has apurinic/apyrimidinic lyase activity (AP lyase activity). A polypeptide having pyrimidine glycosylase activity and AP lyase activity is referred to herein as a "pyrimidine glycosylase/AP lyase," and has "pyrimidine glycosylase/AP lyase activity." Thus, a preferred polypeptide of the present invention has pyrimidine glycosylase/AP lyase activity and a targeting sequence, preferably an exogenous targeting sequence. As used herein, "AP lyase activity" refers to the ability of a polypeptide to catalyze a .beta.-elimination reaction on an abasic site containing DNA, resulting in an .alpha., .beta. unsaturated aldehyde. A polypeptide having pyrimidine glycosylase/AP lyase activity is often referred to in the art as a "pyrimidine dimer specific DNA glycosylase/AP lyase."

Whether a polypeptide has pyrimidine glycosylase/AP lyase activity can be determined by measuring the ability of the polypeptide to incise a target polynucleotide containing damaged bases in the presence of a buffer. The target polynucleotide contains damaged bases, preferably, UV radiation induced pyrimidine dimers. An example of a target polynucleotide is disclosed in the Examples. Preferably, the target polynucleotide is present at a concentration of from about 0.1 nM to about 10 nM. The putative glycosylase/AP lyase is present at a concentration of from about 0.01 nM to about 100 nM. Buffers in which a glycosylase/AP lyase is active are suitable for the assay. Preferably, the buffer includes about 25 mM NaH₂ PO₄. Preferably, the pH is from about 6.5 to about 7.5, more preferably about 6.8. Preferably the buffer contains from about 10 mM NaCl to about 125 mM NaCl, more preferably about 100 mM NaCl. Preferably the buffer contains from about 1 mM EDTA to about 10 mM EDTA, more preferably about 1 mM EDTA. Preferably the buffer contains from about 0.01 mg/mL bovine serum albumin (BSA) to about 1 mg/mL BSA, more preferably about 0.1 mg/mL BSA. Preferably, the temperature of the assay is about 37^oC. The assay can be carried out for at least about 10 seconds to no greater than about 8 hours. Preferably, the assay is about 30 minutes. A polypeptide having pyrimidine glycosylase/AP lyase activity will cause the mobility of the target polynucleotide to change relative to the polynucleotide that has not been exposed to the polypeptide. The polypeptide may be present in a crude cellular extract, preferably isolated, more preferably, purified. Since polypeptides identified in this assay as having pyrimidine glycosylase/AP lyase activity function on UV-irradiated DNAs, these polypeptides identify cyclobutane pyrimidine dimers, and are likely to be active on other UV-induced photoproducts including FapyA and Fapy G.

Individual microbes, preferably Neisseria mucosa and Bacillus sphearicus, and viruses can be screened for the ability to produce polypeptides that have pyrimidine glycosylase activity, preferably pyrimidine glycosylase/AP lyase activity. As used herein, "microbe" refers to prokaryotic organisms. The production by a microbe, or a microbe harboring a virus, of a polypeptide having pyrimidine glycosylase activity, preferably pyrimidine glycosylase/AP lyase activity, can be assayed by, for instance, the ability of the microorganism to incise a target polynucleotide containing damaged bases.

Preferred examples of polypeptides having pyrimidine glycosylase activity include amino acid sequences present in the Chlorella virus isolate PBCV-1 pyrimidine dimer-specific glycosylase (cv-pdg, polypeptide sequence available at Genbank Accession No. AF128160, SEQ ID NO:41), the Bacteriophage T4 pyrimidine dimer-specific glycosylase (T4-pdg, polypeptide sequence available at Genbank Accession No. X04567, SEQ ID NO:42), and the Micrococcus luteus ultraviolet N-glycosylase/AP lyase (Mlu-pdg I, polypeptide sequence available at Genbank Accession No. U22181 , SEQ ID NO:43). Preferably, a polypeptide having pyrimidine glycosylase activity includes amino acid sequences present in cv-pdg (SEQ ID NO:41) or T4-pdg (SEQ ID NO:42).

The present invention further includes polypeptides having pyrimidine glycosylase activity, preferably pyrimidine glycosylase/AP lyase activity, and amino acid identity with the amino acid sequence of SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43, preferably SEQ ID NO:41 or SEQ ID NO:42. Amino acid identity is defined in the context of a comparison between a polypeptide and SEQ ID NO:41 or SEQ ID NO:42, and is determined by aligning the residues of the two amino acid sequences (i.e., a candidate amino acid sequence and the amino acid sequence of SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. A candidate amino acid sequence is the amino acid sequence being compared to an amino acid sequence present in SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43. A candidate amino acid sequence can be isolated from a microbe or a microbe harboring a virus, or can be produced using recombinant techniques, or chemically or enzymatically synthesized. Preferably, two amino acid sequences (i.e., the candidate amino acid sequence and the amino acid sequence present in SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43) are compared using the Blastp program of the BLAST 2 search algorithm, as described by Tatusova, et al. (FEMS Microbiol Lett 1999, 174:247-250), and available at www.ncbi.nlm.nih.gov/gorf/bl2.html. Preferably, the default values for all BLAST 2 search parameters are used, including matrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gap x_dropoff=50, expect=10, wordsize=3, and filter on. In the comparison of two amino acid sequences using the BLAST search algorithm, amino acid identity is referred to as "identities." Preferably, a polypeptide having pyrimidine glycolase activity has an amino acid sequence having, in increasing order of preference, at least about 15% amino acid identity, at least about 30% amino acid identity, at least about 40% amino acid identity, at least about 50% amino acid identity, and most preferably, at least about 60% amino acid identity to SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43.

The polypeptides useful in some aspects of the invention include an active analog or active fragment of SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43. An active analog or active fragment of a pyrimidine glycosylase is one having pyrimidine glycosylase activity, preferably pyrimidine glycosylase/AP lyase activity. Active analogs of a pyrimidine glycosylase include polypeptides having amino acid substitutions that do not eliminate the ability to incise a target polynucleotide containing damaged bases. Substitutes for an amino acid may be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, aspartate, and glutamate. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Examples of preferred conservative substitutions include Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free --OH is maintained; and Gln for Asn to maintain a free NH₂.

Active analogs, as that term is used herein, also include modified polypeptides. Modifications of polypeptides of the invention include chemical and/or enzymatic derivatizations at one or more constituent amino acids, including side chain modifications, backbone modifications, and N- and C-terminal modifications including acetylation, hydroxylation, methylation, amidation, and the attachment of carbohydrate or lipid moieties, cofactors, and the like. Active fragments of a polypeptide include a portion of the polypeptide containing deletions or additions of one or more contiguous or noncontiguous amino acids such that the resulting polypeptide will incise a target polynucleotide containing damaged bases.

The polypeptides of the present invention also include a targeting sequence, preferably, an exogenous targeting sequence. As used herein, a "targeting sequence" is a polypeptide that is fused to a polypeptide having pyrimidine glycosylase activity, preferably pyrimidine glycosylase/AP lyase activity. As used herein, "exogenous targeting sequence" refers to a foreign targeting sequence, i.e., a targeting sequence that is not normally fused to the polypeptide having pyrimidine glycosylase activity, preferably, pyrimidine glycosylase/AP lyase activity. Targeting sequences cause the polypeptide to which they are fused to migrate from the cytoplasm of a cell to an organelle. In one aspect, the targeting sequence is a nuclear localization sequence (NLS) that causes migration into the nucleus. During the transit of the polypeptide that includes an NLS to the nucleus of a cell, the NLS may be cleaved. The invention is not limited by the type of NLS that is fused to the pyrimidine glycosylase, and many NLSs are known to the art (see, for instance, (Moroianu, J. Cell. Biochem. Suppl. 32/33, 76-83 (1999)). An NLS can be present in any location in a polypeptide of the present invention provided the presence of the NLS does not inhibit the pyrimidine glycosylase activity of the polypeptide after the pyrimidine glycosylase is delivered to the nucleus. Preferably, an NLS is present at the carboxy terminal end of a pyrimidine glycosylase. The amino acid sequences of preferred examples of NLSs that can be used in the present invention include a consensus NLS, PKKRKRRL (SEQ ID NO:27) and PKKKRKRL (SEQ ID NO:30).

In another aspect, the targeting sequence is a mitochondria localization sequence (MLS) that causes migration into mitochondria. The invention is not limited by the type of MLS that is fused to the pyrimidine glycosylase. Typically, an MLS is present fused to the amino terminal end of a polypeptide of the present invention. In those aspects of the invention where an MLS is fused to the amino terminal end of a pyrimidine glycosylase, the MLS is cleaved during the transit of the polypeptide that includes the MLS into a cell's mitochondria. In some aspects, the pyrimidine glycosylase, preferably pyrimidine glycosylase/AP lyase, of the present invention are inactive while the MLS is fused, but are active after the MLS is cleaved upon transit into a mitochondrion. Examples of MLSs that can be used include those present in polypeptides that are targeted to the mitochondria, including, for instance, mitochondrial tryphtophanyl-tRNA synthetases (Jorgensen et al., J. Biol. Chem., 275, 16820-16826 (2000)), mitochondrial uracil DNA glycosylase (Otterlei et al., Nucleic Acids Research, 26, 4611-4617 (1998)), manganese superoxide dismutase (Wispe et al., Biochim Biophys Acta, 994, 30-36 (1989)), and ornithine transcarbamylase (Horwich et al., Science 224, 1068-1074 (1984)), among others. Preferred examples of MLSs that can be used in the present invention include MALHSMRKARERWSFIRA (SEQ ID NO:1) and MGVFCLGFWGLGRKLRTFGKGPKQLLSRLCGDHLQ (SEQ ID NO:47).

Whether a polypeptide of the present invention is delivered to the appropriate organelle can be determined by several methods. The polypeptide can be introduced to a eukaryotic cell by, for instance, microinjection of the polypeptide into the cytoplasm of the cell. Alternatively and preferably, the polypeptide is introduced to the cytoplasm of the cell as a composition including the polypeptide and a pharmaceutically acceptable carrier, preferably a liposome, phospholipid, or pH-activated lipid. Pharmaceutically acceptable carriers are described herein. To determine whether the introduced polypeptide is targeted to the nucleus or the mitochodria of a cell, the appropriate organelle can be isolated, and the amount of the polypeptide in the organelle determined. Alternatively and preferably, immunofluorescence analysis with antibody that binds to the polypeptide can be used to determine the intracellular distribution of the polypeptide after it is introduced.

When determining whether a polypeptide of the invention is delivered to the appropriate organelle, the polypeptide may be introduced to the cell as a polynucleotide encoding the polypeptide. The polypeptide is expressed from the polynucleotide and translated in the cytoplasm of the cell. The targeting of the polypeptide to the nucleus or mitochondria of a cell can be determined as described above. It should be noted that as used herein, a polynucleotide encoding the polypeptide is used ex vivo to test whether a polypeptide is delivered to the nucleus or a mitochondrion; polynucleotides are not used for the in vivo delivery of polypeptides of the present invention. Polynucleotides are described herein.

Whether the polypeptide of the present invention retains pyrimidine glycosylase activity, preferably pyrimidine glycosylase/AP lyase activity, once transported into the organelle can be determined by several methods. The polypeptide can be introduced to the cell as described herein, including introduction as a polypeptide and introduction as a polynucleotide that encodes the polypeptide. To measure activity after introduction to the cell, the appropriate organelle can be isolated, the polypeptide isolated from the organelle, and the activity of the isolated polypeptide determined. Alternatively, the repair rate of damaged DNA in the cell can be determined using, for instance, coding sequence-specific repair assays, photoproduct removal, and/or quantitative PCR.

Optionally, a polypeptide of the present invention further includes a series of consecutive amino acids encoding a domain that facilitates the isolation, preferably purification, of the polypeptide. An "isolated" polypeptide or polynucleotide means a polypeptide or polynucleotide that has been either removed from its natural environment, produced using recombinant techniques, or chemically or enzymatically synthesized. Preferably, a polypeptide or polynucleotide of this invention is purified, i.e., essentially free from any other polypeptide or polynucleotide and associated cellular products or other impurities. For instance, domains that are useful in the isolation of a polypeptide that has glycosylase activity, preferably glycosylase/AP lyase activity, include a histidine domain (which can be isolated using nickel-chelating resins), an S-peptide domain (which can be isolated using an S-protein, see Kim, J.-S. et al. Protein Sci 1993 2:348-356), and a chitin binding domain (which can bind to chitin beads, see Chong et al. Gene, 192, 271-281 (1997) and Watanabe et al. J. Bacteriol., 176, 4465-4472 (1994)). Preferably, the domain is present at the carboxy terminal end of the polypeptide. Preferably, the domain can be cleaved from the remainder of the polypeptide (e.g., the polypeptide having pyrimidine glycosylase activity, preferably pyrimidine glycosylase/AP lyase activity, fused to a targeting sequence, preferably an exogenous targeting sequence) by the use of a protease or self-cleaving sequence.

The present invention also provides polynucleotides encoding a polypeptide of the present invention, i.e., a polypeptide having pyrimidine glycosylase activity, preferably, pyrimidine glycosyalse/AP lyase activity, and a targeting sequence, preferably, an exogenous targeting sequence. A polynucleotide may include nucleotide sequences having different functions, including for instance coding sequences, and non-coding sequences such as regulatory sequences. "Coding sequence" and "coding region" are used interchangeably and refer to a polynucleotide that encodes a polypeptide and, when placed under the control of appropriate regulatory sequences expresses the encoded polypeptide. The boundaries of a coding region are generally determined by a translation start codon at its 5' end and a translation stop codon at its 3' end. A regulatory sequence is a nucleotide sequence that regulates expression of a coding region to which it is operably linked. Nonlimiting examples of regulatory sequences include promoters, transcription initiation sites, translation start sites, translation stop sites, and terminators. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A regulatory sequence is "operably linked" to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence.

Polynucleotides encoding a polypeptide of the invention may be obtained from a microbe, preferably Neisseria mucosa and Bacillus sphearicus, or a microbe harboring a virus that produces a polypeptide having pyrimidine glycosylase activity, preferably, pyrimidine glycosylase/AP lyase activity. Methods for isolating a polynucleotide encoding a polypeptide of the invention employs standard cloning techniques known to the art (see, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press (1989) or Ausubel et al., (Eds.) Current Protocols in Molecular Biology, John Wiley & Sons, Inc. New York, N.Y. (1994)).

Preferred examples of polynucleotides include those encoding the Chlorella virus isolate PBCV-1 pyrimidine dimer-specific glycosylase (cv-pdg, nucleotide sequence available at Genbank Accession No. AF128160, SEQ ID NO:44), the Bacteriophage T4 pyrimidine dimer-specific glycosylase (T4-pdg, nucleotides 1777-2193 of Genbank Accession No. X04567, SEQ ID NO:45), and the Micrococcus luteus ultraviolet N-glycosylase/AP lyase (Mlu-pdg I, nucleotides 106-912 of Genbank Accession No. U22181 , SEQ ID NO:46). Preferably, a polynucleotide encoding a polypeptide having pyrimidine glycosylase activity includes the nucleotide sequences encoding cv-pdg (SEQ ID NO:44) or T4-pdg (SEQ ID NO:45).

The present invention further includes polynucleotides encoding polypeptides having pyrimidine glycosylase activity, preferably pyrimidine glycosylase/AP lyase activity, and nucleotide identity with the nucleotide sequence of SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:46, preferably, SEQ ID NO:44 SEQ ID NO:45. Nucleotide identity is defined in the context of a comparison between a polypeptide and SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:46, and is determined by aligning the residues of the two polynucleotides (i.e., the nucleotide sequence of the candidate coding region and the nucleotide sequence of the coding region of SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:46) to optimize the number of identical nucleotides along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of shared nucleotides, although the nucleotides in each sequence must nonetheless remain in their proper order. A candidate coding region is the coding region being compared to a coding region present in SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:46. A candidate nucleotide sequence can be isolated from a microbe or a microbe harboring a virus, or can be produced using recombinant techniques, or chemically or enzymatically synthesized. Preferably, two nucleotide sequences are compared using the Blastn program of the BLAST 2 search algorithm, as described by Tatusova, et al. (FEMS Microbiol Lett 1999, 174:247-250), and available at www.ncbi.nlm.nih.gov/gorf/bl2.html. Preferably, the default values for all BLAST 2 search parameters are used, including reward for match=1, penalty for mismatch=-2, open gap penalty=5, extension gap penalty=2, gap x_dropoff=50, expect=10, wordsize=11, and filter on. In the comparison of two nucleotide sequences using the BLAST search algorithm, structural similarity is referred to as "identities." Preferably, a polynucleotide includes a nucleotide sequence having a structural similarity with the coding region of SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:46 of, in increasing order of preference, at least about 10% identity, at least about 30%, at least about 40% identity, at least about 50% identity, at least about 60% identity, most preferably, at least about 70% identity.

Once a coding region having identity to the coding region present in SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:46, preferably, SEQ ID NO:44 or SEQ ID NO:45, has been identified, the coding region can be isolated and ligated into a vector. A vector is a replicating polynucleotide, such as a plasmid, phage, or cosmid, to which another polynucleotide may be attached so as to bring about the replication of the attached polynucleotide. Construction of vectors containing a polynucleotide of the invention employs standard ligation techniques known in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press (1989) or Ausubel et al., (Eds.) Current Protocols in Molecular Biology, John Wiley & Sons, Inc. New York, N.Y. (1994). A vector can provide for further cloning (amplification of the polynucleotide), i.e., a cloning vector, or for expression of the polypeptide encoded by the coding region, i.e., an expression vector. A vector containing a coding region having identity to the coding region present in SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:46 can be conveniently used to insert the nucleotides encoding a targeting sequence, and optionally, a domain that facilitates the isolation of the encoded polypeptide, in frame with the nucleotides encoding the polypeptide having pyrimidine glycosylase activity, preferably, pyrimidine glycosylase/AP lyase activity. Examples of nucleotides encoding an NLS include CCAAAGAAGAGGAAAAGGAGGCTA (SEQ ID NO:48) and CCAAAGAAAAAGAGGAAGAGGCTA (SEQ ID NO:49). Examples of nucleotides encoding an MLS include

ATGGCGTTACATAGCATGCGCAAAGCGCGCGAACGCTGGAGCTTTATT (SEQ ID NO: 33)

AGAGCA and

ATGGGCGTGTTTTGCTTAGGCCCGTGGGGCTTAGGCCGCAAATTACGC (SEQ ID NO: 34)

ACCCCGGGCAAAGGCCCGTTACAGTTATTATCGCGCTTATGCGGCGAT

CATTTACAG.

The term vector includes, but is not limited to, plasmid vectors, viral vectors, cosmid vectors, or artificial chromosome vectors. Typically, a vector is capable of replication in a bacterial host, for instance E. coli, or in a eukaryotic cell. Preferably the vector is a plasmid.

Selection of a vector depends upon a variety of desired characteristics in the resulting construct, such as a selection marker, vector replication rate, and the like. Suitable host cells for cloning or expressing the vectors herein are prokaryotic or eukaryotic cells. Preferably the host cell secretes minimal amounts of proteolytic enzymes. Suitable prokaryotes include eubacteria, such as gram-negative or gram-positive organisms, for example, E. coli.

An expression vector optionally includes regulatory sequences operably linked to the coding region. The invention is not limited by the use of any particular promoter, and a wide variety are known. Promoters act as regulatory signals that bind RNA polymerase in a cell to initiate transcription of a downstream (3' direction) coding region. The promoter used in the invention can be a constitutive or an inducible promoter. It can be, but need not be, heterologous with respect to the host cell.

Promoter sequences are known for eukaryotes. Most eukaryotic coding regions have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many coding sequences is the CXCAAT region where X may be any nucleotide. At the 3' end of most eukaryotic coding sequences is an AATAAA sequence that may be a signal for addition of the poly A tail to the 3' end of the coding sequence. All these sequences are suitably inserted into eukaryotic expression vectors. The promoter that is normally operably linked to a coding region encoding an polypeptide of the present invention can also be used.

An expression vector can optionally include a ribosome binding site (a Shine Dalgarno site for prokaryotic systems or a Kozak site for eukaryotic systems) and a start site (e.g., the codon ATG) to initiate translation of the transcribed message to produce the enzyme. It can also include a termination sequence to end translation. A termination sequence is typically a codon for which there exists no corresponding aminoacetyl-tRNA, thus ending polypeptide synthesis. The polynucleotide used to transform the host cell can optionally further include a transcription termination sequence. The rrnB terminators, which is a stretch of DNA that contains two terminators, T1 and T2, is an often used terminator that is incorporated into bacterial expression systems. Transcription termination sequences in vectors for eukaryotic cells typically include a polyadenylation signal 3' of the coding region.

Also useful are expression vectors that provide for transient expression in eukaryotic cells of a coding sequence encoding a polypeptide of the invention. In general, transient expression involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of a desired polypeptide encoded by the expression vector. Transient expression systems, including a suitable expression vector and a host cell, allow for the convenient positive identification of polypeptides that are targeted to the appropriate organelle. Methods for the transient expression of coding regions are well known in the art.

The polynucleotide used to transform the host cell optionally includes one or more marker sequences, which typically encode a molecule that inactivates or otherwise detects or is detected by a compound in the growth medium. For example, the inclusion of a marker sequence can render the transformed cell resistant to an antibiotic, or it can confer compound-specific metabolism on the transformed cell. Examples of a marker sequence are sequences that confer resistance to kanamycin, ampicillin, chloramphenicol, tetracycline, and neomycin.

The compositions of the present invention optionally further include a pharmaceutically acceptable carrier. Typically, the composition includes a pharmaceutically acceptable carrier when the composition is used as described below in "Methods of Use." The compositions of the present invention may be formulated in pharmaceutical preparations in a variety of forms adapted to the chosen route of administration. Formulations include those suitable for topical administration, parental administration (for instance intramuscular, intraperitoneal, or intravenous), oral, transdermal, nasal, or aerosol, preferably, topical. Dosages of the compositions of the invention are typically from about 0.01 mg/kg up to about 0.10 mg/kg.

The formulations may be conveniently presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. All methods of preparing a pharmaceutical composition include the step of bringing the active compound (e.g., a polypeptide of the present invention) into association with a carrier that constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulations.

Typically, the compositions of the invention will be administered as needed, typically at least once per day. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the subject treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Preferably, such preparations contain from about 20% to about 80% active compound. The amount of active compound in such therapeutically useful compositions is such that the dosage level will be effective to prevent or suppress the condition the subject has or is at risk for. Such conditions are described hereinbelow.

Preferably, a formulation includes a compound that delivers the active compound to the interior of cells, preferably to the interior of living skin cells under the skin's stratum corneum. Accordingly, such compounds deliver the active compounds across the stratum corneum and then across the outer cellular membrane of living cells. Examples of such compounds include liposomes, phospholipids, and pH-activated lipids (see, for example, U.S. Pat. No. 5,190,762 (Yarosh)).

Formulations suitable for topical administration may include dusting powders, ointments, cremes, gels or sprays for the administration of the active compound to cells, preferably skin cells. Such formulations may optionally include an inorganic pigment, organic pigment, inorganic powder, organic powder, hydrocarbon, silicone, ester, triglyceride, lanolin, wax, cere, animal or vegetable oil, surfactant, polyhydric alcohol, sugar, vitamin, amino acid, antioxidant, free radical scavenger, ultraviolet light blocker, sunscreen agents, preservative, fragrance, thickener, or combinations thereof.

In a particularly preferred embodiment for topical administration, the active compounds of the present invention can be used in cosmetic formulations (e.g., skincare cream, sunscreen, decorative make-up products, and other dermatological compositions) in various pharmaceutical dosage forms, and especially in the form of oil-in-water or water-in-oil emulsions, solutions, gels, or vesicular dispersions. The cosmetic formulations may take the form of a cream which can be applied either to the face or to the scalp and hair, as well as to the human body, in particular those portions of the body that are chronically exposed to sun. They can also serve as a base for a lipstick.

Particularly preferred cosmetic formulations can also include additives such as are usually used in such formulations, for example preservatives, bactericides, perfumes, antifoams, dyes, pigments which have a coloring action, surfactants, thickeners, suspending agents, fillers, moisturizers and/or humectants, fats, oils, waxes or other customary constituents of a cosmetic formulation, such as alcohols, polyols, polymers, foam stabilizers, electrolytes, organic solvents, or silicone derivatives.

Cosmetic formulations typically include a lipid phase and often an aqueous phase. The lipid phase can advantageously be chosen from the following group of substances: mineral oils, mineral waxes oils, such as triglycerides of capric or of caprylic acid, but preferably castor oil; fats, waxes and other natural and synthetic fatty substances, preferably esters of fatty acids with alcohols of low C number, for example with isopropanol, propylene glycol or glycerol, or esters of fatty alcohols with alkanoic acids of low C number or with fatty acids; alkyl benzoates; silicone oils, such as dimethylpolysiloxanes, diethylpolysiloxanes, diphenylpolysiloxanes and mixed forms thereof.

If appropriate, the aqueous phase of the formulations according to the invention advantageously includes alcohols, diols or polyols of low C number and ethers thereof, preferably ethanol, isopropanol, propylene glycol, glycerol, ethylene glycol, ethylene glycol monoethyl or monobutyl ether, propylene glycol monomethyl, monoethyl or monobutyl ether, diethylene glycol monomethyl or monoethyl ether and analogous products, furthermore alcohols of low C number, for example ethanol, isopropanol, 1,2-propanediol and glycerol, and, in particular, one or more thickeners, which can advantageously be chosen from the group consisting of silicon dioxide, aluminium silicates, polysaccharides and derivatives thereof, for example hyaluronic acid, xanthan gum and hydroxypropylmethylcellulose, particularly advantageously from the group consisting of poly-acrylates, preferably a polyacrylate from the group consisting of so-called Carbopols, for example Carbopols of types 980, 981, 1382, 2984 and 5984, in each case individually or in combination.

A preferred cosmetic formulation is a sunscreen composition. A sunscreen can advantageously additionally include at least one further UVA filter and/or at least one further UVB filter and/or at least one inorganic pigment, preferably an inorganic micropigment. The UVB filters can be oil-soluble or water-soluble. Advantageous oil-soluble UVB filter substances are, for example: 3-benzylidenecamphor derivatives, preferably 3-(4-methylbenzylidene)camphor and 3-benzylidenecamphor; 4-aminobenzoic acid derivatives, preferably 2-ethylhexyl 4-(dimethylamino)benzoate and amyl 4-(dimethylamino)benzoate; esters of cinnamic acid, preferably 2-ethylhexyl 4-methoxycinnamate and isopentyl 4-methoxycinnamate; derivatives of benzophenone, preferably 2-hydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxy-4'-methylbenzophenone and 2,2'-dihydroxy-4-methoxybenzophenone; esters of benzalmalonic acid, preferably di(2-ethylhexyl)4-methoxybenzalmalonate. Advantageous water-soluble UVB filter substances are, for example: salts of 2-phenylbenzimidazole-5-sulphonic acid, such as its sodium, potassium or its triethanolammonium salt, and the sulphonic acid itself; sulphonic acid derivatives of benzophenones, preferably 2-hydroxy-4-methoxybenzophenone-5-sulphonic acid and salts thereof; sulphonic acid derivatives of 3-benzylidenecamphor, such as, for example, 4-(2-oxo-3-bornylidenemethyl)benzenesulphonic acid, 2-methyl-5-(2-oxo-3-bornylidenemethyl)benzenesulphonic acid and salts thereof. The list of further UVB filters mentioned which can be used in combination with the active agent(s) according to the invention is not of course intended to be limiting.

Formulations for parenteral administration include a sterile aqueous preparation of the composition, or dispersions of sterile powders that include the composition, which are preferably isotonic with the blood of the recipient. Isotonic agents that can be included in the liquid preparation include sugars, buffers, and sodium chloride. Solutions of the composition can be prepared in water, and optionally mixed with a nontoxic surfactant. Dispersions of the composition can be prepared in water, ethanol, a polyol (such as glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, glycerol esters, and mixtures thereof. The ultimate dosage form is sterile, fluid and stable under the conditions of manufacture and storage. The necessary fluidity can be achieved, for example, by using liposomes, by employing the appropriate particle size in the case of dispersions, or by using surfactants. Sterilization of a liquid preparation can be achieved by any convenient method that preserves the bioactivity of the composition, preferably by filter sterilization. Preferred methods for preparing powders include vacuum drying and freeze drying of the sterile injectable solutions. Subsequent microbial contamination can be prevented using various antimicrobial agents, for example, antibacterial, antiviral and antifungal agents including parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Absorption of the composition by the animal over a prolonged period can be achieved by including agents for delaying, for example, aluminum monostearate and gelatin.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as tablets, troches, capsules, lozenges, wafers, or cachets, each containing a predetermined amount of the active compound as a powder or granules, as liposomes containing the active compound, or as a solution or suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, an emulsion or a draught.

The tablets, troches, pills, capsules, and the like may also contain one or more of the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; an excipient such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, fructose, lactose or aspartame; and a natural or artificial flavoring agent. When the unit dosage form is a capsule, it may further contain a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac, or sugar and the like. A syrup or elixir may contain one or more of a sweetening agent, a preservative such as methyl- or propylparaben, an agent to retard crystallization of the sugar, an agent to increase the solubility of any other ingredient, such as a polyhydric alcohol, for example glycerol or sorbitol, a dye, and flavoring agent. The material used in preparing any unit dosage form is substantially nontoxic in the amounts employed. The active compound may be incorporated into sustained-release preparations and devices.

Methods of Use

The present invention is further directed to methods for treating certain conditions in ex vivo or in vivo cells. The conditions include, for instance, the presence of damaged bases in the cells, preferably skin cells, treating skin cancer, and treating UV induced immunosuppression, and are described in greater detail herein. The cell can be ex vivo or in vivo. As used herein, "ex vivo" refers to a cell that has been removed from the body of an animal. Ex vivo cells include, for instance, primary cells (e.g., cells that have recently been removed from a subject and are capable of limited growth in tissue culture medium), and cultured cells (e.g., cells that are capable of long term culture in tissue culture medium). Such ex vivo methods can be used in various applications, such as determining whether a polypeptide having identity to a polypeptide of the present invention has pyrimidine glycosylase activity, preferably, pyrimidine glycosylase/AP lyase activity. The cell is a eukaryotic cell, preferably, an animal cell, including human, as well as other animals (for instance, mice or rats,) that can be used as animal models in the study of the conditions described herein. Preferably, the cell is a human cell. Cell types that are useful in the methods disclosed herein include cells present in the epidermis, including, for instance, keratinocytes, squamous cells, basal cells, melanocytes, and Langerhans' cells.

Treatment of the conditions described herein can be prophylactic or, alternatively, can be initiated after the development of a condition described herein. Treatment that is prophylactic, for instance, initiated before a subject manifests symptoms of a condition described herein and/or before exposure to an agent that damages DNA, for instance, UV light, oxidative stress, alkylation damage and deamination, preferably UV light, is referred to herein as treatment of a subject that is "at risk" of developing the condition. Accordingly, administration of a composition can be performed before, during, or after the occurrence of the conditions described herein. Treatment initiated after the development of a condition may result in decreasing the severity of the symptoms of one of the conditions, or completely removing the symptoms. Non-limiting examples of subjects particularly suited to receiving the composition are those who may be exposed to natural or artificial UV irradation, individuals having genetic deficiencies in polypeptides involved in DNA repair (for instance, those suffering from xeroderma pigmentosum), and individuals who are immunosuppressed due to disease states (such as AIDS) or transplantation.

A composition that is introduced to a cell, including introduced to a subject, that has or is at risk of developing a condition described herein includes an effective amount of a pyrimidine glycosylase including a targeting sequence. As used herein, an "effective amount" is an amount effective to decrease or prevent (for prophylactic treatment) in a subject the symptoms associated with a condition described herein. Preferably, the composition further includes a pharmaceutically acceptable carrier. Preferably, the composition is administered to the subject by topical administration.

An aspect of the invention is directed to a method for increasing the repair rate of damaged bases in a cell, preferably a skin cell. The method includes introducing to a skin cell exposed to or at risk of exposure to an agent that damages DNA a composition that includes an effective amount of a composition including a pyrimidine glycosylase, preferably a pyrimidine glycosylase/AP lyase, that includes a targeting sequence. The symptoms of this condition include, for instance, the increased presence of damaged DNA, increased mutagenesis rates, increased immunosuppression, increased tumor formation (for instance, increased actinic keratosis, increased basal cell carcinoma, and increased squamous cell carcinoma, and possibly increased melanoma), and increased incidence of apoptotic cells.

Whether the repair rate of damaged bases in a cell is increased can be determined by, for instance, assaying for the amount of damaged DNA in cells using a variety of techniques including coding sequence-specific repair assays (Bohr et al., Cell, 40, 359-369 (1985)), and photoproduct removal as determined by ELISA assays using antibodies directed against cis-syn dimers (Clarkson et al., Mutation Res., 112, 287-299 (1983)). Alternatively, when human cells are used, the removal of lesions can be assayed by quantitative PCR assay that is specific for human mitochondrial DNA (see Balleinger et al., Exp. Eye Res., 68, 765-772 (1999), and Ballinger et al., Circ, Res., 86, 960-966 (2000)). For instance, ex vivo cells can be exposed to an agent that damages DNA, preferably UV light, and treated with a composition including a polypeptide of the present invention. After a period of time sufficient to allow repair, the amount of damaged DNA in the cells can be determined and compared to the same type of cell that was not treated with the polypeptide. The presence of less damaged DNA in the cell treated with the polypeptide relative to the cell not treated indicates the polypeptide increases the repair rate of DNA. The repair rate of damaged DNA in in vivo cells may also be determined. For instance, an animal can be exposed to an agent that damages DNA, and treated with a composition including a polypeptide of the present invention. After a period of time sufficient to allow repair, skin biopsies are prepared and the amount of damaged DNA determined and compared to skin biopsies obtained from animals not treated with the polypeptide. The presence of less damaged DNA in cells in the biopsies treated with the polypeptide relative to cells in the biopsies not treated indicates the polypeptide increases the repair rate of DNA. Commonly accepted in vivo models are available for testing whether a polypeptide will increase the repair rate of DNA (for human models, see, for instance, Yarosh et al., Photochem. Photobiol., 69, 136-140 (1999); for animal models, see, for instance, Mitchell et al., J. Invest. Dermatol., 95, 55-59 (1990)).

The present invention further provides methods for treating mutagenesis in a cell, preferably a skin cell, in response to an agent that damages DNA, preferably UV light. In this aspect of the invention, mutagenesis rates are decreased. Mutagenesis results when repair of damaged DNA does not occur and, upon replication of the DNA, a different base is inserted. The method includes introducing to a skin cell exposed to or at risk of exposure to an agent that damages DNA a composition that includes an effective amount of a pyrimidine glycosylase, preferably a pyrimidine glycosylase/AP lyase, that includes a targeting sequence. Whether the rate of mutagenesis in a cell is reduced can be determined by, for instance, hprt mutagenesis assays (O'Neill et al, Mutat. Res., 45, 103-109 (1977)). Briefly, the measurement of mutagenesis using an hprt assay involves the selection of mammalian cells that are resistant to the killing effects of 6-thioguanine through a mutation in the hprt coding sequence. The assay relies on an inability of hprt-cells to activate 6-thioguanine for incorporation into DNA that results in cell killing. All cells with wild type hprt are killed upon 6-thioguanine selection. The cells can be in vivo or ex vivo. The rate of mutagenesis in cells treated with a polypeptide of the present invention can be determined and compared to the rate of mutagenesis in cells not treated. The presence of a lower mutagenesis rate in treated cells relative to untreated cells indicates the polypeptide decreases the mutagenesis rate of DNA.

Also provided by the present invention are methods for treating immunosuppression in a cell, preferably a skin cell, in response to an agent that damages DNA, preferably UV light. The presence of damaged DNA results in a temporary, reversible immunosuppression. The method includes introducing to a skin cell exposed to or at risk of exposure to an agent that damages DNA a composition that includes an effective amount of a pyrimidine glycosylase, preferably a pyrimidine glycosylase/AP lyase, that includes a targeting sequence. Whether immunosuppression in response to a DNA damaging agent is decreased can be determined by, for instance, measuring the transcription and/or translation of coding sequences that promote immunosuppression in response to a DNA damaging agent. For instance, the transcription and/or translation of a coding sequence encoding interleukin-10 (IL-10) or tumor necrosis factor alpha (TNFa) can be measured using Northern blot analyses or commercially available antibody kits. The immunosuppression in cells treated with a polypeptide of the present invention can be determined and compared to the immunosuppression in cells not treated. The presence of higher levels of IL-10 and/or TNFa in treated cells relative to untreated cells indicates the polypeptide decreases the immunosuppression of a cell in response to agents that damage DNA.

The present invention is also directed to methods for treating tumor formation in a cell, preferably a skin cell, in response to an agent that damages DNA, preferably UV light. In this aspect of the invention, tumor formation is decreased. The types of tumors that may occur in response to an agent that damages DNA include actinic keratosis, basal cell carcinoma, squamous cell carcinoma, and melanoma. The method includes introducing to a skin cell that is at risk of developing a tumor in response to an agent that damages DNA a composition that includes an effective amount of a pyrimidine glycosylase, preferably a pyrimidine glycosylase/AP lyase, that includes a targeting sequence. Cells at risk of developing a tumor in response to an agent that damages DNA include cells exposed to or at risk of exposure to an agent that damages DNA. Whether the formation of tumors in an animal is reduced can be determined by the use of animal models, for instance mice, that have been exposed to solar simulated light or exposure to sunlight. Solar simulated light is light having a spectral profile which is similar to natural solar irradiation, i.e. the emission spectrum of a solar simulator looks similar to spectrum of a solar noon day. Wavelengths of light include .about.295-400 nm so is inclusive of UVA, UVB but not UVC which does not get through the ozone (see, for instance, Yoon et al., J. Mol. Biol., 299, 681-693 (2000). The presence of a tumor can be determined by methods known in the art, and typically include cytological and morphological evalution. The cells can be in vivo or ex vivo, including obtained from a biopsy. The rate of tumor formation in cells treated with a polypeptide of the present invention can be determined and compared to the rate of mutagenesis in cells not treated. The presence of a lower rates of tumor formation in treated cells relative to untreated cells indicates the polypeptide decreases tumor formation.

Another aspect of the present invention is directed to treating the formation of apoptotic cells, preferably apoptotic skin cells, in response to an agent that damages DNA, preferably UV light. Apoptotic cells are cells undergoing, or that have undergone, programmed cell death. In this aspect of the invention, the formation of apoptotic cells is decreased. The method includes introducing to a skin cell exposed to or at risk of exposure to an agent that damages DNA a composition that includes an effective amount of a pyrimidine glycosylase, preferably a pyrimidine glycosylase/AP lyase, that includes a targeting sequence. Whether the formation of apoptotic cells is reduced can be determined by, for instance, assays that detect apoptotic cells. Such assays include immunohistochemistry using antibodies against apoptotic-specific polypeptides associated with apoptotic cells, including, for instance, anti-caspase 8, anti-procaspase 9, and anti-G3PDH antibodies. Such antibodies are known to the art, and are available from, for instance, Trevigan (Gaithersberg, Md.) and Sigma (St. Louis, Mo.). The cells can be in vivo or ex vivo, including obtained from a biopsy. The formation of apoptotic cells in cells treated with a polypeptide of the present invention can be determined and compared to the formation of apoptitic cells in cells not treated. The presence of a lower apoptosis rate in treated cells relative to untreated cells indicates the polypeptide decreases the formation of apoptotic cells.

Claim 1 of 7 Claims

What is claimed is:

1. An isolated polypeptide comprising:

an amino acid sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43; and

a nuclear or mitochondrial targeting sequence.

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