Human DNA ligase III

Details Human DNA ligase III Human DNA ligase III

1998-04-03

2001-09-04

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

C536S023200

Reexamination Certificate

active

06284504

ABSTRACT:

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. The polypeptide of the present invention has been putatively identified as Human DNA Ligase III. The invention also relates to inhibiting the action of such polypeptides.
DNA strand breaks and gaps are generated transiently during replication, repair and recombination. In mammalian cell nuclei, rejoining of such strand breaks depends on several different DNA polymerases and DNA ligase enzymes.
The mechanism for joining of DNA strand interruptions by DNA ligase enzymes has been widely described. The reaction is initiated by the formation of a covalent enzyme-adenylate complex. Mammalian and viral DNA ligase enzymes employ ATP as cofactor, whereas bacterial DNA ligase enzymes 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 &egr;-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 ligase enzymes, but is more short lived than the adenylylated enzyme. In the final step of DNA ligation, unadenylylated DNA ligase enzymes required for the generation of a phosphodiester bond catalyze displacement of the AMP residue through attack by the adjacent 3′-hydroxyl group on the adenylylated site.
The occurrence of three different DNA ligase enzymes, DNA Ligase I, II and III, was established previously by biochemical and immunological characterization of purified enzymes (Tomkinson, A. E. et al., J. Biol. Chem., 266:21728-21735 (1991) and Roberts, E., et al., J. Biol. Chem., 269:3789-3792 (1994)). However, the inter-relationship between these proteins was unclear as a cDNA clone has only been available for DNA Ligase I, the major enzyme of this type in proliferating cells (Barnes, D. E., et al., PNAS USA, 87:6679-6683 (1990)). The main function of DNA Ligase I appears to be the joining of Okazaki fragments during lagging-strand DNA replication (Waga, S., et al., J. Biol. Chem. 269:10923-10934 (1994); Li, C., et al., Nucl. Acids Res., 22:632-638 (1994); and Prigent, C., et al., Mol. Cell. Biol., 14:310-317 (1994)).
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.,
Eur. 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 ligase enzymes. The active site lysine residue is located at position 568. 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 II and III in being unable to ligate oligo (dT) with a poly (rA) complementary strand.
Human DNA Ligase III is more firmly associated with the cell nuclei. This enzyme is a labile protein, which is rapidly inactivated at 42° C. DNA Ligase III resembles other eukaryotic DNA Ligase enzymes in requiring ATP as cofactor, but the enzyme differs from DNA Ligase I in having a higher association for ATP. DNA Ligase III 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)).
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 super-coiled DNA (Elder, R. H. et al.,
Eur. J. Biochem
., 203:53-58 (1992)).
Clues as to the physiological role of DNA Ligase III have come from its physical interaction in a high salt-resistant complex with another nuclear protein, the XRCC1 gene product (Caldecott, K. W., et al.,
Mol. Cell. Biol
., 14:68-76 (1994) and Ljungquist, S., et al.,
Mutat. Res
., 314:177-186 (1994)). The XRCC1 gene encodes a 70 kDa protein, that by itself does not appear to join DNA strand breaks (Caldecott, K. W., et al.,
Mol. Cell. Biol
., 14:68-76 (1994); Ljungquist, S., et al.,
Mutat. Res
., 314:177-186 (1994) and Thompson, L. H., et al.,
Mol. Cell. Biol
., 10:6160-6171 (1990)). However, mutant rodent cells deficient in XRCC1 protein exhibit reduced DNA Ligase III activity, defective strand break repair, an anomalously high level of sister chromatid exchanges, are hyper-sensitive to simple alkylating agents and ionizing radiation, and have an altered mutation spectrum after exposure to ethyl methanesulfonate (Caldecott, K. W., et al.,
Mol. Cell. Biol
., 14:68-76 (1994); Ljungquist, S., et al.,
Mutat. Res
., 314:177-186 (1994); Thompson, L. H., et al.,
Mol. Cell. Biol
., 10:6160-6171 (1990); and Op het Veld, C. W., et al.,
Cancer Res
., 54:3001-3006 (1994)). These data indicate that XRCC1 mutant cells are defective in base excision-repair, and strongly suggest that both DNA Ligase III and XRCC1 are active in this process (Dianov, G., and Lindahl, T.,
Curr. Biol
., 4:1069-1076 (1994)).
A purified mammalian protein fraction active in repair and recombination processes in vitro was shown to contain a ligase with the properties of Human DNA Ligase III, but no detectable amounts of Human DNA Ligase I (Jessberger, R., et al.,
J. Biol. Chem
., 268:15070-15079 (1993)). The role of the distinct enzyme, DNA Ligase II, remains unclear, although an observed increase in DNA Ligase II activity during meiotic prophase suggests a role in meiotic recombination (Higashitani, A., et al.,
Cell Struct. Funct
., 15:67-72 (1990)). Comparison of
32
P-adenylylated DNA Ligase II and III by partial or complete proteolytic cleavage patterns indicated that these two enzymes share extensive amino acid sequence similarity or identity flanking their active sites, but that they are quite different from DNA Ligase I (Roberts, E., et al.,
J. Biol. Chem
., 269:3789-3792 (1994)). Neither DNA Ligase I, II nor III is exclusively a mitochondrial enzyme.
The polynucleotide of the present invention and polypeptide encoded thereby have been putatively identified as human DNA Ligase III as a result of size, amino acid sequence homology to DNA Ligase II and ability to bind XRCC1 protein. Heretofore, the gene sequence of DNA Ligase III was not known.
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

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