EP0796320A1 - Human dna ligase iii - Google Patents

Abstract

A human DNA Ligase III polypeptide and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptide via gene therapy for the treatment of disorders associated with a defect in DNA Ligase III. Antagonists against such polypeptides and their use as a therapeutic to destroy unwanted cells are also disclosed. Diagnostic assays to detect mutant DNA Ligase III genes are also disclosed.

Description

HUMAN DNA LIGASE III

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotideε, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is Human DNA Ligase III. The invention also relates to inhibiting the action of such polypeptides.

DNA strand interruptions and gaps are generated during replication, repair and recombination. In mammalian cell nuclei, rejoining of such breaks depends on several different DNA polymeraseε and DNA ligases. The occurrence of three different DNA ligases was established previously by biochemical and immunological characterization of purified enzymes (Tomkinson, A.E., et al., J. Biol. Chem., 266:21728- 21735 (1991) ) . DNA ligases are enzymes that catalyze DNA replication, excision repair and recombinational repair in mammalian cells (Li, J.J. and Kelly, T.J. , PNAS. USA, 81:6973-77 (1984) and Wook, R.O. et al . , Cell. 53:97-106 (1988)). In bacteria, three DNA ligases, namely DNA Ligase I, DNA Ligase II and DNA Ligase III have been discovered, while in humans all three have been found but only DNA Ligase I has been cloned.

-ι- A full-length human cDNA encoding DNA Ligase I has been obtained by functional complementation of a S. cereviasiae cdc9 temperature-sensitive DNA ligase mutant (Barker, D.G., Bur. J. Biochem.. 162:659-67 (1987)). The full-length cDNA encodes a 102-kDa protein of 919 amino acid residues. There is no marked sequence homology to other known proteins except for microbial DNA ligases. The active site lysine residue is located at position 568. DNA Ligase I requires magnesium and ATP for activity. The main function of DNA Ligase I is the joining of Okazaki fragments during lagging-strand DNA replication. It also effectively seals single-strand breaks in DNA and joins restriction enzyme DNA fragments with staggered ends. The enzyme is also able to catalyze blunt- end joining of DNA. DNA Ligase I can join oligo (dT) molecules hydrogen-bonded to poly (dA) , but the enzyme differs from T4 DNA Ligase in being unable to ligate oligo (dT) with a poly (rA) complementary strand.

Human DNA Ligase II is more firmly associated with the cell nuclei. This enzyme is a labile protein, which is rapidly inactivated at 42°C. DNA Ligase II resembles other eukaryotic DNA Ligases in requiring ATP as cof ctor, but the enzyme differs from DNA Ligase I in having a much higher association for ATP. DNA Ligase II catalyzes the formation of phosphodiester bonds with an oligo (dT) • poly (rA) substrate, but not with an oligo (rA) • poly (dT) substrate, so it differs completely from DNA Ligase I in this regard (Arrand, J.E. et al., J. Biol. Chem.. 261:9079-82 (1986)).

A recently detected enzyme, which is larger than DNA Ligase II and apparently unrelated to that protein, has been named DNA Ligase III (Tomkinson, A.E. et al., J. Biol. Che . , 266:21728-35 (1991)) . DNA Ligase III resembles DNA Ligase I, and differs from DNA Ligase II, in binding only weakly to hydroxylapatite in having a low affinity, for ATP. DNA Ligase I and III however are not closely related. DNA Ligase III repairs single strand breaks in DNA efficiently, but it is unable to perform either blunt-end joining or AMP- dependent relaxation of supercoiled DNA (Elder, R.H. et al . , Bur. J. Biochem.. 203:53-58 (1992)) .

The mechanism for joining of DNA strand interruptions by DNA ligases has been widely described. The reaction is initiated by the formation of a covalent enzyme-adenylate complex. Mammalian and viral DNA ligases employ ATP as cofactor, whereas bacterial DNA ligases use NAD to generate the adenylyl group. The ATP is cleaved to AMP and pyrophosphate with the adenylyl residue linked by a phosphoramidate bond to the e-amino group of a specific lysine residue at the active site of the protein (Gumport, R.I., et al . , PNAS, 68:2559-63 (1971)). Reactivated AMP residue of the DNA ligase-adenylate intermediate is transferred to the 5' phosphate terminus of a single strand break in double stranded DNA to generate a covalent DNA-AMP complex with a 5'-5' phosphoanhydride bond. This reaction intermediate has also been isolated for microbial and mammalian DNA ligases, but is more short lived than the adenylylated enzyme. In the final step of DNA ligation, unadenylylated DNA ligases required for the generation of a phosphodiester bond catalyzes displacement of the AMP residue through attack by the adjacent 3'-hydroxyl group on the adenylylated site.

The polypeptide of the present invention has been putatively identified as a human DNA Ligase III as a result of amino acid sequence homology.

In accordance with one aspect of the present invention, there are provided novel mature polypeptides which are human DNA Ligase III, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.

In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding human DNA Ligase III, including mRNAs, DNAs, cDNAs, genomic DNAs as well as analogs and biologically active and diagnostically or therapeutically useful fragments thereof.

In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a human DNA Ligase III nucleic acid sequence, under conditions promoting expression of said protein and subsequent recovery of said protein.

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors.

In accordance with another aspect of the present invention there is provided a method of treating conditions which are related to insufficient human DNA Ligase III activity via gene therapy comprising inserting the DNA Ligase III gene into a patient's cells either in vivo or ex vivo. The gene is expressed in transduced cells and as a result, the protein encoded by the gene may be used therapeutically, for example, to prevent disorders associated with defects in DNA, for example, abnormal cellular proliferation, for example tumors, to treat severe immunosuppression, stunted growth and lymphσma, as well as cellular hypersensitivity to DNA-damaging agents.

In accordance with yet a further aspect of the present invention, there is also provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to human sequences which may be used diagnostically to detect a mutation in the gene encoding DNA Ligase III.

In accordance with yet another aspect of the present invention, there are provided antagonists to such polypeptides, which may be manufactured intracellularly or administered through gene therapy to inhibit the action of such polypeptides, for example, to target and destroy undesired cells, e.g., cancer cells.

These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

Figure 1 shows the cDNA sequence and the corresponding deduced amino sequence of the DNA Ligase III polypeptide. The standard one letter abbreviation for amino acids is used.

Figure 2 illustrates the amino acid homology between human DNA Ligase I (upper line)and human DNA Ligase III (lower line) .

Figure 3 is a copy of a gel illustrating the presence of DNA Ligase III in different human tissues. Panel A is an autoradiograph of Northern analysis of the human DNA Ligase III gene and panel B is an ethidium bromide stained gel as a loading reference. The results show that human DNA Ligase III is mainly expressed in the thymus, testis and heart.

In accordance with an aspect of the present invention, there is provided an isolated nucleic acid (polynucleotide) which encodes for the mature polypeptide having the deduced amino acid sequence of Figure 1 or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. 75880 on August 31, 1994.

A polynucleotide encoding a polypeptide of the present invention may be obtained from testis, thymus and heart. The polynucleotide of this invention was discovered in a cDNA library derived from human activated T-cells. It is structurally related to the DNA ligase family. It contains an open reading frame encoding a protein of 899 amino acid residues. The protein exhibits the highest degree of homology to rabbit DNA ligase with 29 % identity and 51 % similarity over a the entire protein. It is also important that there is a conserved active lysine residue which is bordered on either side by a hydrophobic amino acid residue, and the sequence E-KYDG-R is common to enzymes from different sources such as mammalian cells, yeasts, vaccinia virus and bacteriophage T7.

The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA of Figure 1 or the deposited cDNA.

The polynucleotide which encodes for the mature polypeptide of Figure 1 or for the mature polypeptide encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non- coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.

Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.

The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone. The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure 1 or of the coding sequence of the deposited clone. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.

The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention. The marker sequence may be a hexa- histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 50% and preferably 70% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides . As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNA of Figure 1 or the deposited cDNA.

The deposit(s) referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. These deposits are provided merely as convenience to those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. §112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.

The present invention further relates to a DNA Ligase III polypeptide which has the deduced amino acid sequence of Figure 1 or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide.

The terms "fragment," "derivative" and "analog" when referring to the polypeptide of Figure 1 or that encoded by the deposited cDNA, means a polypeptide which retains essentially the same biological function or activity as such polypeptide. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of Figure 1 or that encoded by the deposited cDNA may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol) , or (iv) one in which the additional amino acids are fused to the mature polypeptide, which is employed for purification of the mature polypeptide. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) . For example, a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the DNA Ligase III genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids,- vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(ε) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp. the phage lambda P_L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.

As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli. Streptomyces, Salmonella tvphimurium; fungal cells, such as yeast; insect cells such as Drosophila and Sf9; animal cells such as CHO, COS or Bowes melanoma; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen) , pBS, pDIO, phagescript, psiX174, pbluescript SK, pbskε, pNH8A, pNH16a, pNHlβA, pNH46A (Stratagene) ; ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia) . However, any other plasmid or vector may be used as long as they are replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are PKK232-8 and PCM7. Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P_R, P_L and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation. (Davis, L. , Dibner, M. , Battey, I., Basic Methods in Molecular Biology, (1986) ) .

The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers. Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting treinsformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRPl gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK) , o;-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis. Salmonella tvphimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017) . Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA) . These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.

Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981) , and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

The DNA Ligase III polypeptide can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) . Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.

The DNA Ligase III polypeptides and agonists and antagonists which are polypeptides, described below, may be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as "gene therapy."

Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.

Once the DNA Ligase III polypeptide is being expressed intra-cellularly via gene therapy, it may be used to repair single-strand breaks in DNA which result from DNA-damaging agents, e.g., UV radiation. Several human syndromes result from autosomal recessive inheritance for the DNA ligase gene. These syndromes cause severe immunodeficiency and greatly increases the susceptibility of abnormal cellular differentiation due to the disrepair of DNA while at the cellular level they are characterized by chromosome instability and hypersensitivity to DNA-damaging agents. These syndromes include Fanconi's anemia and Blackfan-diamond anemia.

The polypeptide of the present invention may also be employed to treat severe immunosuppression which is the result of a defect in the DNA ligase III gene and stunted growth and lymphoma which results from defective rejoining of DNA.

Similarly, the polynucleotide of the present invention is also useful for identifying other molecules which have similar biological activity. An example of a screen for this is isolating the coding region of the DNA Ligase III gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.

The polypeptide and/or polynucleotide of the present invention may also be employed in relation to scientific research, synthesis of DNA and for the manufacture of DNA vectors. The polypeptide and/or polynucleotide of the present invention may be sold into the research market. Thus, for example DNA Ligase III may be used for ligation of DNA sequences in viϋrσin a manner similar to other DNA ligases of the art.

This invention also provides a method of screening compounds to identify those which enhance (agonists) or block (antagonists) the DNA-joining reaction catalyzed by human DNA Ligase III . An example of such method comprises combining ATP and DNA Ligase III and DNA having single-strand breaks with the compound under conditions where the DNA ligase would normally cleave ATP to AMP and the AMP is transferred to the 5' phosphate terminus of a single strand break in double- stranded DNA to generate a covalent DNA-AMP complex with the single strand break being subsequently repaired. The DNA having the single-strand breaks may be supplied in the above example by mutant cells which are deficient in proteins that are responsible for strand break repair, for example mutant rodent cells deficient in XRCCl and the cdc9 S. Cerevisiae DNA ligase mutant. The ability of the compound to enhance or block the catalysis of this reaction could then be measured to determine if the compound is an effective agonist or antagonist.

Human DNA Ligase III is produced and functions intra- cellulary, therefore, any antagonists must be intra-cellular. Potential antagonists to human DNA Ligase III include antibodies which are produced intra-cellularly. For example, an antibody identified as antagonizing DNA Ligase III may be produced intra-cellularly as a single chain antibody by procedures known in the art, such as transforming the appropriate cells with DNA encoding the sigle chain antibody to prevent the function of human DNA Ligase III.

Another potential antagonist is an an isense construct prepared using antisense technology. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are baβed on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix - see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al. , Science, 251: 1360 (1991) ) , thereby preventing transcription and the production of DNA Ligase III. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the DNA Ligase III (antisense - Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of DNA Ligase III.

Yet another potential antagonist includes a mutated form, or mutein, of DNA Ligase III which recognizes DNA but does not repair single-strand breaks and, therefore, acts to prevent human DNA Ligase III from functioning.

The antagonists may be employed to target undesired cells, e.g., cancer cells, since the prevention of DNA Ligase III prevents repair of single-strand breaks in DNA and will eventually result in death of the cell.

The small molecule agonists and antagonists of the present invention may be employed in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the molecule and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the pharmaceutical compositions of the present 96/14394 PCMJS94/12922 invention may be employed in conjunction with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenient manner such as by the oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The pharmaceutical compositions are administered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, they are administered in an amount of at least about 10 μg/kg body weight and in most cases they will be administered in an amount not in excess of about 8 mg/Kg body weight per day. In most cases, the dosage is from about 10 μg/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.

Fragments of the full length Human DNA Ligase III gene may be used as a hybridization probe for a cDNA library to isolate the full length DNA Ligase III gene and to isolate other genes which have a high sequence similarity to the DNA Ligase III gene. Probes of this type can be, for example, 30, 40, 50 75, 90, 100 or 150 bases. Preferably, however, the probes have between 30 and 50 base pairs. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions, exons, and introns. The probe may be labelled, for example, by radioactivity to facilitate identification of hypbridization.

This invention also provides the use of the human DNA Ligase III gene as a diagnostic. For example, some diseases result from inherited defective genes. These genes can be detected by comparing the sequence of the defective gene with that of a normal one. That is, a mutant gene would be associated with hypersensitivity to DNA-damaging agents and an elevated susceptibility to abnormal cell growth, for example tumors and cancer. Individuals carrying mutations in the human DNA Ligase III gene may be detected at the DNA level by a variety of techniques. Nucleic acids used for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al . , Nature, 324:163-166 (1986) prior to analysis. RNA or cDNA may also be used for the same purpose. Deletions or insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled DNA Ligase III RNA or alternatively, radiolabeled DNA Ligase III antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.

Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing fornamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase protection and SI protection or the chemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)) .

Thus, the detection of a specific DNA sequence may be achieved by method such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing, or the use of restriction enzymes, e.g., restriction fragment length polymorphisms, and Southern blotting of genomic DNA. Also, mutations may be detected by in situ analysis.

In addition, some diseases are a result of, or are characterized by, changes in gene expression which can be detected by changes in the mRNA. Alternatively, the DNA Ligase III gene can be used as a reference to identify individuals expressing a decreased level of DNA Ligase III protein, e.g., by Northern blotting.

The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the cDNA is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.

Fluorescence in si tu hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 500 or 600 bases,* however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. FISH requires use of the clones from which the EST was derived, and the longer the better. For example, 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time. For a review of this technique, see Verma et al. , Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988) .

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library) . The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes) . The gene of the present invention has been mapped to chromosome 13q33-34.

Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.

With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes l megabase mapping resolution and one gene per 20 kb) .

The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) .

Techniques described for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention.

The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight.

In order to facilitate understanding of the following examples certain frequently occurring methods and/or terms will be described.

"Plasmids" are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer, incubation times of about l hour at 37"C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res., 8:4057 (1980) .

"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatiε, T. , et al., Id., p. 146) . Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated.

Unless otherwise stated, transformation was performed as described in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973) .

Bxample l Bacterial Expression and Purification of DNA Liσase III

The DNA sequence encoding DNA Ligase III, ATCC # 75880 is initially amplified using PCR oligonucleotide primers corresponding to the 5' sequences of the processed DNA Ligase III protein. The 5' oligonucleotide primer has the sequence 5' GCGGGATCCATGAGACTAATTCTTCCTCAG 3' contains a Bam HI restriction enzyme site (underlined) followed by 21 nucleotides of DNA Ligase III coding εequence εtarting from the presumed terminal amino acid of the processed protein codon. The 3' sequence 5' GCGCTGCAGTTAAATCAAATACTGG'rri G 3' contains complementary sequences to a Pst I site (underlined) and is followed by 21 nucleotides of DNA Ligase III at C- terminal of DNA Ligase III. The restriction enzyme siteε correspond to the restriction enzyme siteε on the bacterial expression vector pQE-9 (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, CA, 91311) . pQE-9 encodes antibiotic resistance (Amp^r) , a bacterial origin of replication (ori) , an IPTG- regulatable promoter operator (P/O) , a ribosome binding site (RBS) , a 6-His tag and restriction enzyme sites. pQE-9 is then digested with Bam HI and Pst I. The amplified sequences are ligated into pQE-9 and inserted in frame with the sequence encoding for the histidine tag and the RBS. The ligation mixture is then used to transform E. coli strain Ml5/rep 4 available from Qiagen under the trademark Ml5/rep 4 by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989) . M15/rep4 contains multiple copies of the plasmid pREP4, which expresses the lad repressor and also confers kanamycin resistance (Kan^r) . Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis. Clones containing the desired constructs are grown overnight (0/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) . The O/N culture is uεed to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto pyranoside") is then added to a final concentration of 1 mM. IPTG induces by inactivating the la repressor, clearing the P/0 leading to increased gene expression. Cells are grown an extra 3 to 4 hours. Cells are then harvested by centrifugation. The cell pellet iε εolubilized in the chaotropic agent 6 Molar Guanidine HCl. After clarification, εolubilized protein extract is purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography 411:177-184 (1984) ) and eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, OOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized) . After incubation in this solution for 12 hours the protein is dialyzed to 10 mmolar sodium phosphate.

Example 2 Clonino and expression of DNA Liσase III using the baculovirus expression system

A DNA sequence encoding full length DNA Ligase III protein, ATCC # 75880, is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:

The 5' primer has the sequence 5' GCGCCCGGGATGAGACTAATT CTTCTCCAG 3' and contains a Sma I restriction enzyme site (in bold) followed first by 21 nucleotides resembling an efficient signal for the initiation of tranεlation in eukaryotic cells (Kozak, M. , J. Mol. Biol., 196:947-950, (1987) ) (the initiation codon for tranεlation "ATG" iε underlined) .

The 3' primer haε the sequence 5' G∞GGTAC ITAAATCAAATACTGGTTTTC 3'and contains the cleavage εite for the restriction endonuclease Asp 718 (in bold) and 21 nucleotides complementary to the C-terminal sequence of the DNA Ligase III gene. The amplified sequences were isolated from a 1% agarose gel uεing a commercially available kit ("Geneclean, " BIO 101 Inc., La Jolla, Ca.) . The fragment waε then digeεted with the endonucleases Sma I and Asp 718 and then purified again on a 1% agarose gel. This fragment is designated F2.

The vector pRGl (modification of pVL941 vector, discussed below) iε used for the expresεion of the DNA Ligase III protein using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555) . This expression vector contains the strong polyhedrin promoter of the Autographa califomica nuclear polyhedrosis virus (AcMNPV) followed by the recognition siteε for the restriction endonucleases Sma I and Asp 718. The polyadenylation site of the simian virus (SV)40 iε used for efficient polyadenylation. For an easy selection of recombinant viruses the beta-galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sideε by viral sequences for the cell-mediated homologous recombination of cotransfected wild-type viral DNA. Many other baculovirus vectors could be used in place of pRGl such as pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D., Virology, 170:31-39).

The plasmid iε digested with the restriction enzymes Sma I and Asp 718 and then dephosphorylated uεing calf intestinal phosphatase by procedures known in the art. The DNA is then isolated from a 1% agarose gel using the commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.). This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNA ligase. E.coli HB101 cells are then transformed and bacteria identified that contained the plasmid (pBac DNA Ligase III) with the DNA Ligase III gene using the enzymes Sma I and Asp 718. The sequence of the cloned fragment is confirmed by DNA sequencing.

5 μg of the plasmid pBac DNA Ligase III was cotransfected with 1.0 μg of a commercially available linearized baculovirus ("BaculoGold™ baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Feigner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417

(1987)) . lμg of BaculoGold™ virus DNA and 5 μg of the plasmid pBac DNA Ligase III are mixed in a sterile well of a microtiter plate containing 50 μl of serum free Grace's medium (Life Technologies Inc., Gaitherεburg, MD) . Afterwards 10 μl Lipofectin pluε 90 μl Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the tranεfection mixture iε added dropwiεe to the Sf9 inεect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1ml Grace' medium without serum. The plate is rocked back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27°C. After 5 hours the transfection solution is removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The plate iε put back into an incubator and cultivation continued at 27°C for four dayε.

After four days the supernatant is collected and a plaque assay performed similar as described by Summers and Smith (supra) . As a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) iε uεed which allows an easy isolation of blue stained plaques. (A detailed description of a "plaque assay" can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologieε Inc., Gaitherεburg, page 9- 10) .

Four dayε after the serial dilution of the viruεes is added to the cells, blue stained plaques are picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruses is then resuspended in an Eppendorf tube containing 200 μl of Grace's medium. The agar is removed by a brief centrifugation and the supernatant containing the recombinant baculoviruεes is used to infect Sf9 cells seeded in 35 mm disheε. Four dayε later the εupernatants of these culture dishes are harvested and then stored at 4°C. Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus V-DNA Ligase III at a multiplicity of infection (MOD of 2. Six hours later the medium iε removed and replaced with SF900 II medium minus methionine and cysteine (Life Technologies Inc., Gaitherεburg). 42 hours later 5 μCi of ³⁵S-methionine and 5 μCi ^3SS cysteine (Amersham) are added. The cells are further incubated for 16 hours before they are harvested by centrifugation and the labelled proteins visualized by SDS-PAGE and autoradiography.

Example 3 Expression of Recombinant DNA Liσase III in COS cells

The expression of plasmid, DNA Ligase III HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) atπpicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site. A DNA fragment encoding the entire DNA Ligase III precursor and a HA tag fused in frame to its 3' end was cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. The HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H. Ni an, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, Cell 37:767 (1984)). The infusion of HA tag to our target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence encoding DNA Ligase III, ATCC # 75880, is constructed by PCR on the original EST cloned uεing two primerε: the 5' primer 5' GCGGAATTCATGAGACTAA'1'rCl'rCCTCAG 3' containε an Eco RI site (underlined) followed by 21 nucleotides of DNA Ligase III coding sequence starting from the initiation codon; the 3' sequence 5'GCGCTCGAGTCAAGCGTAG TCTGGGACGTCGTATGGGTAAATCAAATACTGGTTTTGTTC 3' contains complementary sequences to an Xho I site (underlined) , translation stop codon, HA tag and the last 21 nucleotides of the DNA Ligase III coding sequence (not including the stop codon) . Therefore, the PCR product containε an Eco RI εite, DNA Ligaεe III coding sequence followed by HA tag fused in frame, a translation termination stop codon next to the HA tag, and an Xho I site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with Eco RI and Xho I restriction enzyme and ligated. The ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037) the transformed culture is plated on ampicillin media plates and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment. For expression of the recombinant DNA Ligase III, COS cells are transfected with the expression vector by DEAE- DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatiε, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)) . The expression of the DNA Ligase III HA protein iε detected by radiolabelling and i munoprecipitation method (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Preεε, (1988)) . Cells are labelled for 8 hours with ³⁵S-cysteine two days post transfection. Culture media are then collected and cells are lyεed with detergent (RIPA buffer (150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Triε, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate and culture media are precipitated with a HA εpecific monoclonal antibody. Proteinε precipitated are analyzed on 15% SDS-PAGE gelε. Example 4 Expresεion pattern of DNA Ligase III in human tiεεue

Northern blot analyεiε may be performed to examine the levels of expreεεion of DNA Ligaεe III in human tissues. Total cellular RNA samples are isolated with RNAzol™ B system (Biotecx Laboratories, Inc. 6023 South Loop East, Houston, TX 77033) . About 15μg of total RNA isolated from each human tissue specified is separated on 1% agarose gel and blotted onto a nylon filter (Sambrook, Fritsch, and Maniatis, Molecular Cloning, Cold Spring Harbor Presε, (1989)). The labeling reaction iε done according to the Stratagene Prime- It kit with 50ng DNA fragment. The labeled DNA iε purified with a Select-G-50 column (5 Prime - 3 Prime, Inc. 5603 Arapahoe Road, Boulder, CO 80303) . The filter containing the particular RNA blot is then hybridized with radioactive labeled full length DNA Ligase III gene at 1,000,000 cpm/ml in 0.5 M NaP0₄, pH 7.4 and 7% SDS overnight at 65"C. After wash twice at room temperature and twice at 60"C with 0.5 x SSC, 0.1% SDS, the filter is then exposed at -70"C overnight with an intensifying screen. The message RNA for DNA Ligase III is abundant in the thymus, testis and heart (see Figure 3) .

Numerous modifications and variations of the present invention are posεible in light of the above teachingε and, therefore, within the εcope of the appended claims, the invention may be practiced otherwise than as particularly described.

SEQUENCE LISTING

(1) GENERAL INFORMATION: (i) APPLICANT: WEI, Y.

(ii) TITLE OF INVENTION: Human DNA Ligase III

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN,

CECCHI, STEWART _ OLSTEIN

(B) STREET: 6 BECKER FARM ROAD

(C) CITY: ROSELAND

(D) STATE: NEW JERSEY

(E) COUNTRY: USA

(F) ZIP: 07068

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5 INCH DISKETTE

(B) COMPUTER: IBM PS/2

(C) OPERATING SYSTEM: MS-DOS

(D) SOFTWARE: WORD PERFECT 5.1

(Vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: Submitted herewith

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA

(A) APPLICATION NUMBER:

(B) FILING DATE: (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: FERRARO, GREGORY D.

(B) REGISTRATION NUMBER: 36,134

(C) REFERENCE/DOCKET NUMBER: 325800-229

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 201-994-1700

(B) TELEFAX: 201-994-1744

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 3325 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: CDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

CCACAGCGCT GTAGACTGCG CCGCATTAGA AGCCTGGCCT CCTGATGCTG TGCTCTTCAT 60

CTJM5ACCCAA GCCCCAGGTC GTGGGACGAT TTCTCCCGTT TT GACTCCC TGGAACTGTA 120

TTGCCTGCTT TACCTGCGTA CATGTTGATT CTTTCTCATG GCAACCCCGC AGGAAACCAT 180

CAAGATCTCA TTTTACAGCT GGGATTCTCT GGTTCACAGA GGTAACGGAG CTTGCCCGAG 240

GCCAGTTAAA CGAGAAGATT CATCACCGCT TTGATGGCTG CCTCACAAAC TTCACAAACT 300

GTTGCATCTC ACGTTCCTTT TCGAGATTTG TGTTCAACTT TAGAACGAAT ACAGAAAAGT 360

AAAGGACGTG CAGAAAAAAT CAGACACTTC AGGGAATTTT TAGATTCTTG GAGAAAATTT 420

CATGATGCTC TTCATAAGAA CCACAAAGAT GTCACAGACT CTTTTTATCC AGCAATGAGA 480

CTAATCTTC CTCAGCTAGA AAGAGAGAGA ATGGCCTATG GAATTAAAGA AACTATGCTT 540

GCTAAGCTTT ATATTGAGTT GCTTAATTTA CCTAGAGATG GAAAAGATGC CCTCAAACTT 600

TTAAACTACA GAACACCCAC TGGAACTCAT GGAGATGCTG GAGACTTTGC AATGATTGCA 660

TATTTTGTGT TGAAGCCAAG ATGTTTACAG AAAGGAAGTT TAACCATACA GCAAGTAAAC 720

GACCTTTTAG ACTCAATTGC CAGCAATAAT TCTGCTAAAA GAAAAGACCT AATAAAAAAG 780

AGCCTTCTTC AACTTATAAC TCAGAGTTCA GCACTTGAGC AAAAGTGGCT TATACGGATG 840

ATCATAAAGG ATTTAAAGCT TGGTGTTAGT CAGCAAACTA TCTTTTCTGT TTTTCATAAT 900

GATGCTGCTG AGTTGCATAA TGTCACTACA GATCTGGAAA AAGTCTGTAG GCAACTGCAT 960

GATCCTTCTG TAGGACTCAG TGATATTTCT ATCACTTTAT TTTCTGCATC AAAACCAATG 1020

CTAGCTGCTA TTGCAGATAT TGAGCACATT GAGAAGGATA TGAAACATCA GAGTTTCTAC 1080

ATAGAAACCA AGCTAGATGG TGAACGTATG CAAATGCACA AAGATGGAGA TGTATATAAA 1140 TACTTCTCTC GAAATGGATA TAACTACACT GATCAGTTTG GTGCTTCTCC TACTGAAGGT 1200 TCTCTTACCC CATTCATTCA TAATGCATTC AAAGCAGATA TACAAATCTG TATTCTTGAT 1260 GGTGAGATGA TGGCCTATAA TCCTAATACA CAAACTTTCA TGCAAAAGGG AACTAAGTTT 1320 GATATTAAAA GAATGGTAGA GGATTCTGAT CTGCAAACTT GTTATTGTGT TTTTGATGTA 1380 TTGATGGTTA ATAATAAAAA GCTAGGGCAT GAGACTCTGA GAAAGAGGTA TGAGATCTT 1440 AGTAGTATTT TTACACCAAT TCCAGGTAGA ATAGAAATAG TGCAGAAAAC ACAAGCTCAT 1500 ACTAAGAATG AAGTAATTGA TGCATTGAAT GAAGCAATAG ATAAAAGAGA AGAGGGAATT 1560 ATGGTAAAAC AACCTCTATC CATCTACAAG CCAGACAAAA GAGGTGAAGG GTGGTTAAAA 1620 ATTAAACCAG AGTATGTCAG TGGACTAATG GATGAATTGG ACATTTTAAT TGTTGGAGGA 1680 TATTGGGGTA AAGGATCACG GGGTGGAATG ATGTCTCATT TTCTGTGTGC AGTAGCAGAG 1740 AAGCCCCCTC GTGGTGAGAA GCCATCTGTG TTTCATACTC TCTCTCGTGT TGGGTCTGGC 1800 TGCACCATGA AAGAACTGTA TGATCTGGGT TTGAAATTGG CCAAGTATTG GAAGCCTTTT 1860 CATAGAAAAG CTCCACCAAG CAGCATTTTA TGTGGAACAG AGAAGCCAGA AGTATACATT 1920 GAACCTTGTA ATTCTGTCAT TGTTCAGATT AAAGCAGCAG AGATCGTACC CAGTGATATG 1980 TATAAAACTG GCTGCACCTT GCGTTTTCCA CGAATTGAAA AGATAAGAGA TGACAAGGAG 2040 TGGCATGAGT GCATGACCCT GGACGACCTA GAACAACTTA GGGGGAAGGC ATCTGGTAAG 2100 CTCGCATCTA AACACCTTTA TATAGGTGGT GATGATGAAC CACAAGAAAA AAAGCGGAAA 2160 GCTGCCCCAA AGATGAAGAA AGTTATTGGA ATTATTGAGA ACTTAAAAGC ACCTAACCTT 2220 AACTAACGTA ACAAAATTTC TAATATATTT GAAGATGTAG AGTTTTGTGT TATGAGTGGA 2280 ACAGATAGCC AGCCAAAGCC TGACCTGGAG AACAGAATTG CAGAATTTGG TGGTTATATA 2340 GTACAAAATC CAGGCCCAGA CACGTACTGT GTAATTGCAG GGTCTGAGAA CATCAGAGTG 2400 AAAAACATAA TTTTGTCAAA TAAACATGAT GTTGTCAAGC CTGCATGGCT TTTAGAATGT 2460 TTTAAGACCA AAAGCTTTGT ACCATGGCAG CCTCGCTTTA TGATTCATAT GTGCCCATCA 2520 ACCAAAGAAC ATTTTGCCCG TGAATATGAT TGCTATGGTG ATAGTTATTT CATTGATACA 2580 GACTTGAACC AACTGAAGGA AGTATTCTCA GGAATTAAAA ATTCTAACGA GCAGACTCCT 2640 GAAGAAATGG CTTCTCTGAT TGCTGATTTA GAATATCGGT ATTCCTGGGA TTGCTCTCCT 2700 CTCAGTATGT TTCGACGCCA CACCGTTTAT TTGGACTCGT ATGCTGTTAT TAATGACCTG 2760 AGTACCAAAA ATGAGGGGAC AAGGTTAGCT ATTAAAGCCT TGGAGCTTCG GTTTCATGGA 2820 GCAAAAGTAG TTTCTTGTTT AGCTGAGGGA GTGTCTCATG TAATAATTGG GGAAGATCAT 2880 AGTCGTGTTG CAGATTTTAA AGCTTTTAGA AGAACTTTTA AGAGAAAGTT TAAAATCCTA 2940 AAAGAAAGTT GGGTAACTGA TTCAATAGAC AAGTGTGAAT TACAAGAAGA AAACCAGTAT 3000 TTGATTTAAA GCTAGGTTTC CTAGTGAGGA AAGCCTCTGA TCTGGCAGAC TCATTGCAGC 3060 AGGTGGTAAT GATAAAATAC TAAACTACAT TTTATTTTTG TATCTTAAAA ATCTATGCCT 3120 AAAAAGTATC ATTACATATA GGAAAACAAT AATTTTAACT TTTAAGGTTG AAAAGACAAT 3180 AGCCCAAAGC CAAGAAAGAA AAATTATCTT GAATGTAGTA TTCAATGATT TTTTATGATC 3240 AAGGTGAAAT AAACAGTCTA AAGAAGAGGT GTTTTTATAA TATCCATATA GAAATCTAGA 3300 ATTTTTACTT AGATACTAAT AAAAT 3325

(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 910 AMINO ACIDS

(B) TYPE: AMINO ACID (C) STRANDEDNESS :

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: PROTEIN

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Ala Ala Ser Gin Thr Ser Gin Thr Val Ala Ser Hiε Val Pro

5 10 15

Phe Ala Asp Lys Cys Ser Thr Leu Glu Arg lie Gin Lyε Ser Lyε

20 25 30

Gly Arg Ala Glu Lyε lie Arg Hiε Phe Arg Glu Phe Leu Aεp Ser

35 40 45

Trp Arg Lyε Phe His Asp Ala Leu His Lys Asn His Lys Asp Val

50 55 60

Thr Asp Ser Phe Tyr Pro Ala Met Arg Leu lie Leu Pro Gin Leu

65 70 75

Glu Arg Glu Arg Met Ala Tyr Gly lie Lys Glu Thr Met Leu Ala

80 85 90

Lyε Leu Tyr lie Glu Leu Leu Aεn Leu Pro Arg Aεp Gly Lyε Asp

95 100 105

Ala Leu Lyε Leu Leu Aεn Tyr Arg Thr Pro Thr Gly Thr Hiε Gly

110 115 120

Aεp Ala Gly Asp Phe Ala Met lie Ala Tyr Phe Val Leu Lys Pro

125 130 135

Arg Cys Leu Gin Lys Gly Ser Leu Thr lie Gin Gin Val Asn Asp

140 145 150

Leu Leu Asp Ser lie Ala Ser Asn Asn Ser Ala Lys Arg Lyε Asp

155 160 165

Leu lie Lys Lys Ser Leu Leu Gin Leu lie Thr Gin Ser Ser Ala

170 175 180

Leu Glu Gin Lys Trp Leu lie Arg Met lie lie Lys Asp Leu Lys

185 190 195

Leu Gly Val Ser Gin Gin Thr lie Phe Ser Val Phe His Asn Asp

200 205 210 Ala Ala Glu Leu Hiε Aεn Val Thr Thr Aεp Leu Glu Lyε Val Cys

215 220 225

Arg Gin Leu His Asp Pro Ser Val Gly Leu Ser Asp lie Ser lie

230 235 240

Thr Leu Phe Ser Ala Ser Lys Pro Met Leu Ala Ala lie Ala Asp

245 250 255 lie Glu His lie Glu Lyε Aεp Met Lyε Hiε Gin Ser Phe Tyr lie

260 265 270

Glu Thr Lyε Leu Asp Gly Glu Arg Met Gin Met Hiε Lys Asp Gly

275 280 285

Asp Val Tyr Lys Tyr Phe Ser Arg Asn Gly Tyr Asn Tyr Thr Asp

290 295 300

Gin Phe Gly Ala Ser Pro Thr Glu Gly Ser Leu Thr Pro Phe lie

305 310 315

His Asn Ala Phe Lys Ala Asp lie Gin lie Cys lie Leu Aεp Gly

320 325 330

Glu Met Met Ala Tyr Asn Pro Asn Thr Gin Thr Phe Met Gin Lys

335 340 345

Gly Thr Lys Phe Asp lie Lys Arg Met Val Glu Asp Ser Asp Leu

350 355 360

Gin Thr Cys Tyr Cys Val Phe Asp Val Leu Met Val Asn Asn Lyε

365 370 375

Lys Leu Gly His Glu Thr Leu Arg Lys Arg Tyr Glu lie Leu Ser

380 385 390

Ser lie Phe Thr Pro lie Pro Gly Arg lie Glu lie Val Gin Lyε

395 400 405

Thr Gin Ala His Thr Lys Asn Glu Val lie Asp Ala Leu Asn Glu

410 415 420

Ala lie Asp Lys Arg Glu Glu Gly lie Met Val Lys Gin Pro Leu

425 430 435

Ser lie Tyr Lys Pro Asp Lys Arg Gly Glu Gly Trp Leu Lys lie

440 445 450

Lys Pro Glu Tyr Val Ser Gly Leu Met Asp Glu Leu Aεp lie Leu

455 460 465 lie Val Gly Gly Tyr Trp Gly Lyε Gly Ser Arg Gly Gly Met Met

470 475 480

Ser Hiε Phe Leu Cyε Ala Val Ala Glu Lyε Pro Pro Pro Gly Glu

485 490 495

Lys Pro Ser Val Phe His Thr Leu Ser Arg Val Gly Ser Gly Cys

500 505 510

Thr Met Lys Glu Leu Tyr Asp Leu Gly Leu Lys Leu Ala Lyε Tyr

515 520 525

Trp Lyε Pro Phe Hiε Arg Lyε Ala Pro Pro Ser Ser lie Leu Cyε

530 535 540

Gly Thr Glu Lys Pro Glu Val Tyr lie Glu Pro Cys Asn Ser Val

545 550 555

He Val Gin He Lys Ala Ala Glu He Val Pro Ser Asp Met Tyr

560 565 570

Lys Thr Gly Cyε Thr Leu Arg Phe Pro Arg He Glu Lyε He Arg

575 580 585

Asp Asp Lys Glu Trp Hiε Glu Cyε Met Thr Leu Asp Asp Leu Glu

590 595 600

Gin Leu Arg Gly Lyε Ala Ser Gly Lyε Leu Ala Ser Lyε Hiε Leu

605 610 615

Tyr He Gly Gly Asp Asp Glu Pro Gin Glu Lys Lys Arg Lys Ala

620 625 630

Ala Pro Lys Met Lyε Lyε Val He Gly He He Glu Hiε Leu Lyε

635 640 645

Ala Pro Asn Leu Thr Asn Val Asn Lys He Ser Asn He Phe Glu

650 655 660

Asp Val Glu Phe Cys Val Met Ser Gly Thr Asp Ser Gin Pro Lys

665 670 675

Pro Aεp Leu Glu Aεn Arg He Ala Glu Phe Gly Gly Tyr He Val

680 685 690

Gin Aεn Pro Gly Pro Aεp Thr Tyr Cyε Val He Ala Gly Ser Glu

695 700 705

Asn He Arg Val Lys Aεn He He Leu Ser Asn Lys His Asp Val

710 715 720 Val Lyε Pro Ala Trp Leu Leu Glu Cyε Phe Lyε Thr Lyε Ser Phe

725 730 735

Val Pro Trp Gin Pro Arg Phe Met He Hiε Met Cys Pro Ser Thr

740 745 750

Lys Glu Hiε Phe Ala Arg Glu Tyr Asp Cys Tyr Gly Aεp Ser Tyr

755 760 765

Phe He Asp Thr Asp Leu Asn Gin Leu Lys Glu Val Phe Ser Gly

770 775 780 He Lyε Aεn Ser Asn Glu Gin Thr Pro Glu Glu Met Ala Ser Leu

785 790 795

He Ala Asp Leu Glu Tyr Arg Tyr Ser Trp Asp Cyε Ser Pro Leu

800 805 810 Set Met Phe Arg Arg His Thr Val Tyr Leu Asp Ser Tyr Ala Val

815 820 825

He Asn Asp Leu Ser Thr Lys Asn Glu Gly Thr Arg Leu Ala He

830 835 840

Lys Ala Leu Glu Leu Arg Phe His Gly Ala Lys Val Val Ser Cyε

845 850 855

Leu Ala Glu Gly Val Ser His Val He He Gly Glu Asp His Ser

860 865 870

Arg Val Ala Aεp Phe Lyε Ala Phe Arg Arg Thr Phe Lyε Arg Lyε

875 880 885

Phe Lyε He Leu Lyε Glu Ser Trp Val Thr Aεp Ser He Aεp Lyε

890 895 900

Cys Glu Leu Gin Glu Glu Asn Gin Tyr Leu He

905 910

Claims

WHAT IS CLAIMED IS:

1. An isolated polynucleotide selected from the group consisting of:

(a) a polynucleotide encoding the polypeptide having the deduced amino acid sequence of Figure 1 or a fragment, analog or derivative of said polypeptide;

(b) a polynucleotide encoding the polypeptide having the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. 75880 or a fragment, analog or derivative of said polypeptide.

2. The polynucleotide of Claim 1 wherein the polynucleotide is DNA.

3. The polynucleotide of Claim 1 wherein the polynucleotide is RNA.

4. The polynucleotide of Claim l wherein the polynucleotide is genomic DNA.

5. The polynucleotide of Claim 2 wherein said polynucleotide encodes the polypeptide having the deduced amino acid sequence of Figure l.

6. The polynucleotide of Claim 2 wherein said polynucleotide encodes the polypeptide encoded by the cDNA of ATCC Depoεit No. 75880.

7. The polynucleotide of Claim 1 having the coding sequence of the polypeptide as shown in Figure 1.

8. The polynucleotide of Claim 2 having the coding sequence of the polypeptide deposited as ATCC Deposit No. 75880.

9. A vector containing the DNA of Claim 2.

10. A host cell genetically engineered with the vector of Claim 9.

11. A procesε for producing a polypeptide compriεing: expressing from the host cell of Claim 10 the polypeptide encoded by said DNA.

12. A process for producing cells capable of expresεing a polypeptide compriεing genetically engineering cellε with the vector of Claim 9.

13. An isolated DNA hybridizable to the DNA of Claim 2 and encoding a polypeptide having DNA Ligase III activity.

14. A polypeptide selected from the group consisting of (i) a polypeptide having the deduced amino acid sequence of Figure 1 and fragments, analogs and derivatives thereof and (ii) a polypeptide encoded by the cDNA of ATCC Deposit No. 75880 and fragments, analogs and derivatives of said polypeptide.

15. The polypeptide of Claim 14 wherein the polypeptide has the deduced amino acid sequence of Figure 1.

16. An antibody against the polypeptide of claim 14.

17. An antagonist against the polypeptide of claim 14.

18. A method for the treatment of a patient having need of DNA Ligase III activity comprising: - adminiεtering to the patient a therapeutically effective amount of the polypeptide of claim 14 by providing to the patient DNA encoding εaid polypeptide and expreεεing εaid polypeptide in vivo.

19. A method for the treatment of a patient having need to inhibit DNA Ligase III compriεing: adminiεtering to the patient a therapeutically effective amount of the antagonist of Claim 17.

20. The method of claim 19 wherein said antagonist is administered by providing to the patient DNA encoding said antagonist and expressing said antagonist in vivo.

21. A method for identifying antagoniεtε and agoniεtε compriεing: combining DNA Ligase III, DNA having single- strand breaks and a compound to be screened under conditionε where the single-strand break would be repaired by the DNA Ligase III; and determining if the compound enhances or blocks the repair.

22. A method for diagnosing abnormal cellular proliferation or a susceptibility to abnormal cellular proliferation in a patient comprising: detecting in a sample derived from a patient a nutation in the nucleic acid sequence of claim 1.